High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.

Deciphering patterns of connectivity between neurons in the brain is a critical step toward understanding brain function. Imaging-based neuroanatomical tracing identifies area-to-area or sparse neuron-to-neuron connectivity patterns, but with limited throughput. Barcode-based connectomics maps large numbers of single-neuron projections, but remains a challenge for jointly analyzing single-cell transcriptomics. Here, we established a rAAV2-retro barcode-based multiplexed tracing method that simultaneously characterizes the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets and found that projection-defined vmPFC neurons are molecularly heterogeneous. We identified transcriptional signatures of projection-specific vmPFC neurons, and verified Pou3f1 as a marker gene enriched in neurons projecting to the lateral hypothalamus, denoting a distinct subset with collateral projections to both dorsomedial striatum and lateral hypothalamus. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.

Mapping of clonal lineages across developmental stages in human neural differentiation.

The cell lineages across developmental stages remain to be elucidated. Here, we developed single-cell split barcoding (SISBAR) that allows clonal tracking of single-cell transcriptomes across stages in an in vitro model of human ventral midbrain-hindbrain differentiation. We developed "potential-spective" and "origin-spective" analyses to investigate the cross-stage lineage relationships and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. We uncovered many previously uncharacterized converging and diverging trajectories. Furthermore, we demonstrate that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multilineage fates of a progenitor cell-type represent the collective results of distinct rather than similar clonal fates of individual progenitors, each with distinct molecular signatures. Specifically, we uncovered a ventral midbrain progenitor cluster as the common clonal origin of midbrain dopaminergic (mDA) neurons, midbrain glutamatergic neurons, and vascular and leptomeningeal cells and identified a surface marker that can improve graft outcomes.

PEDOT/PVC-modified amperometric carbon electrodes for acetylcholine detection.

We present here new amperometric electrodes for the selective and quantitative detection of acetylcholine. The detection was achieved based on the electrodeposition of a carbon electrode with poly(3,4-ethylenedioxythiophene) (PEDOT) followed by the drop-casting of an ionophore-doped poly(vinyl) chloride (PVC) membrane. This work paves the way for future applied research to study neurological disorders.

Fast generation of forebrain oligodendrocyte spheroids from human embryonic stem cells by transcription factors.

Oligodendrocyte spheroids (OL-spheroids) containing oligodendrocytes and neurons provide an accessible system to dissect demyelinating diseases and test therapeutic treatment. However, generation of human OL-spheroids is still technically challenging and time-consuming until now. Here, we presented evidence that overexpression of SOX10 and OLIG2 (SO) in human embryonic stem cells (hESCs)-derived ventral forebrain neural progenitors is sufficient to produce forebrain pre-oligodendrocytes (pre-OLs) and mature oligodendrocytes (OLs) within 20-40 days. More importantly, optimizing this procedure by overexpression of SO in ventral forebrain spheroids, we successfully generated OL-spheroids with pre-OLs, mature OLs, and neurons 40 days after OL-induction. We further demonstrated oligodendrocyte-neuron interactions and obvious axon myelination in OL-spheroids. Finally, over 30% cells developed into mature oligodendrocytes with forebrain identity and myelinate axons in mouse brain 3 months after transplantation. This study provides a strategy to generate forebrain OL-spheroids rapidly and efficiently which would facilitate development of new therapeutics for demyelinating disorders.

Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcomes in a Parkinson's disease model.

Human pluripotent stem cell-based (hPSC-based) replacement therapy holds great promise for the treatment of Parkinson's disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we have characterized the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including the ventral midbrain, the isthmus, and the ventral hindbrain, resulting in a heterogenous donor cell population. We reconstructed the differentiation trajectory of the mDA lineage and identified calsyntenin 2 (CLSTN2) and protein tyrosine phosphatase receptor type O (PTPRO) as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate high enrichment of mDA neurons (up to 80%) following progenitor cell sorting and transplantation. Marker-sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker-sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.

Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite.

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

In situ construction of hierarchical Co/MnO@graphite carbon composites for highly supercapacitive and OER electrocatalytic performances.

The development of new electrode materials with high specific capacity for excellent supercapacitive storage and energy conversion is highly desirable. The combination of metal and metal oxide with carbon is an effective strategy to achieve active bimetallic nanocatalysts. Herein, we developed a facile method to synthesize CoxMn1-xO@GC and Co/MnO@GC nanocomposites by an in situ conversion of Co-Mn PBAs. The as-prepared carbon hybrids, especially the resulting Co/MnO@GC carbonized under 700 °C (Co/MnO@GC-700), preserve the nanocubic morphology of Co-Mn PBAs and show excellent supercapacitance and OER performance. Specifically, an outstanding specific capacitance of 2275 F g-1 can be obtained with Co/MnO@GC-700 as the electrode material at a current density of 4 A g-1. When used as OER catalysts, Co/MnO@GC-700 shows a low overpotential of only 358 mV at 10 mA cm-2 in 1 M KOH. Moreover, a fabricated asymmetric supercapacitor device (ASC device), in combination with active carbon, shows a high cell voltage of 1.7 V and a considerably high specific capacitance of 246 F g-1 at 2 A g-1. Our nanoarchitecture design derived from PBAs provides a new opportunity for future applications in high-performance energy storage and transformation systems.

Delicate Control of Multishelled Zn-Mn-O Hollow Microspheres as a High-Performance Anode for Lithium-Ion Batteries.

Mixed/composite oxides of transition metals with hollow structures, especially multishelled hollow architecture, have promising potential for different applications, but their syntheses still remain a big challenge. Herein, a facile coordination polymer precursor method was developed to construct various multishelled Zn-Mn-O hollow microspheres, including ZnMnO3, ZnMn2O4, and ZnMn2O4/Mn2O3. The composition of the hollow structures can be adjusted by controlling the composition of the coordination polymer precursors, which are easily obtained with Zn2+, Mn2+, and salicylic acid under solvothermal conditions. With a simple programmable heating process, the shell of the hollow structures can be adjusted and double-/triple-shelled ZnMnO3, ZnMn2O4, and ZnMn2O4/Mn2O3 hollow microspheres have been controllably obtained. When the triple-shelled ZnMn2O4 hollow microspheres are used as anode materials for lithium-ion batteries, excellent activity and enhanced stability can be achieved. The triple-shelled hollow ZnMn2O4 exhibits a reversible capacity of 537 mA·h·g-1 at 400 mA·g-1 and a nearly 100% capacity retention after 150 cycles. This strategy is facile and scalable for the production of high-quality complex hollow nanostructures, with the possibility of extension to the preparation of other mixed metal oxides with complex structures.