大熊 真人

The direct reprogramming of cells has tremendous potential in in vitro neurological studies. Previous attempts to convert blood cells into induced neurons have presented several challenges, necessitating a less invasive, efficient, rapid, and convenient approach. The current study introduces an optimized method for converting somatic cells into neurons using a nonsurgical approach that employs peripheral blood cells as an alternative source to fibroblasts. We have demonstrated the efficacy of a unique combination of transcription factors, including NEUROD1, and four Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4, and c-MYC), in generating glutamatergic neurons within 3 wk. This approach, which requires only five pivotal factors (NEUROD1, OCT3/4, SOX2, KLF4, and c-MYC), has the potential to create functional neurons and circumvents the need for induced pluripotent stem cell (iPSC) intermediates, as evidenced by single-cell RNA sequencing and whole-genome bisulfite sequencing, along with lineage-tracing experiments using Cre-LoxP system. While fibroblasts have been widely used for neuronal reprogramming, our findings suggest that peripheral blood cells offer a potential alternative, particularly in contexts where minimally invasive sampling and procedures convenient for patients are emphasized. This method provides a rapid strategy for modeling neuronal diseases and contributes to advancements in drug discovery and personalized medicine.

When exposed to X-rays, scintillators emit visible luminescence. X-ray-mediated optogenetics employs scintillators for remotely activating light-sensitive proteins in biological tissue through X-ray irradiation. This approach offers advantages over traditional optogenetics, allowing for deeper tissue penetration and wireless control. Here, we assessed the short-term safety and efficacy of candidate scintillator materials for neuronal control. Our analyses revealed that lead-free halide scintillators, such as Cs3Cu2I5, exhibited significant cytotoxicity within 24 h and induced neuroinflammatory effects when injected into the mouse brain. In contrast, cerium-doped gadolinium aluminum gallium garnet (Ce:GAGG) nanoparticles showed no detectable cytotoxicity within the same period, and injection into the mouse brain did not lead to observable neuroinflammation over four weeks. Electrophysiological recordings in the cerebral cortex of awake mice showed that X-ray-induced radioluminescence from Ce:GAGG nanoparticles reliably activated 45% of the neuronal population surrounding the implanted particles, a significantly higher activation rate than europium-doped GAGG (Eu:GAGG) microparticles, which activated only 10% of neurons. Furthermore, we established the cell-type specificity of this technique by using Ce:GAGG nanoparticles to selectively stimulate midbrain dopamine neurons. This technique was applied to freely behaving mice, allowing for wireless modulation of place preference behavior mediated by midbrain dopamine neurons. These findings highlight the unique suitability of Ce:GAGG nanoparticles for X-ray-mediated optogenetics. The deep tissue penetration, short-term safety, wireless neuronal control, and cell-type specificity of this system offer exciting possibilities for diverse neuroscience applications and therapeutic interventions.

Body movements influence brain-wide neuronal activities. In the sensory cortex, thalamocortical bottom-up inputs and motor-sensory top-down inputs are thought to affect the dynamics of membrane potentials (V_m) of neurons and change their processing of sensory information during movements. However, direct perturbation of the axons projecting to the sensory cortex from other remote areas during movements has remained unassessed, and therefore the interareal circuits generating motor-related signals in sensory cortices remain unclear. Using a G_i-coupled opsin, eOPN3, we here inhibited interareal signals incoming to the whisker primary somatosensory barrel cortex (wS1) of awake male mice and tested their effects on whisking-related changes in neuronal activities in wS1. Spontaneous whisking in air induced the changes in spike rates of a fraction of wS1 neurons, which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of V_m. Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and V_mdynamics of wS1 neurons during whisking. In contrast, inhibition of axons from the whisker-related thalamus (wTLM) and the whisker secondary somatosensory cortex (wS2) to wS1 largely attenuated the whisking-related supra- and sub-threshold V_mdynamics of wS1 neurons. Notably, silencing inputs from wTLM markedly decreased the modulation depth of whisking phase-tuned neurons, while inhibiting wS2 inputs did not impact the whisking variable tuning of wS1 neurons. Thus, sensorimotor integration in wS1 during spontaneous whisking is predominantly facilitated by direct synaptic inputs from wTLM and wS2 rather than from wM1. Significance statementThe traditional viewpoint underscores the importance of motor-sensory projections in shaping movement-induced neuronal activity within sensory cortices. However, this study challenges such established views. We reveal that the synaptic inputs from the whisker primary motor cortex do not alter the activity patterns and membrane potential dynamics of neurons in the whisker primary somatosensory cortex (wS1) during spontaneous whisker movements. Furthermore, we make a novel observation that inhibiting inputs from the whisker secondary somatosensory cortex (wS2) substantially curtails movement-related activities in wS1, leaving the tuning to whisking variables unaffected. These findings provoke a reconsideration of the role of motor-sensory projections in sensorimotor integration and bring to light a new function for wS2-to-wS1 projections.

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

<title>Abstract</title> Gonadal hormones function as neurosteroids in the retina; however, their targets in the retina have not yet been identified. The present study examined the effects of gonadal hormones on glutamatergic circuits in the retina. Extracellular glutamate concentrations, which correspond to the amount of glutamate released, were monitored using an enzyme-linked fluorescent assay system. Progesterone and pregnenolone both increased extracellular glutamate concentrations at a physiological concentration in pregnancy, whereas estrogen and testosterone did not. Synaptic level observations using a patch clamp technique revealed that progesterone increased the activity of glutamatergic synapses. We also investigated whether high concentrations of gonadal hormones induced changes in the retina during pregnancy. The present results indicate that progesterone activates glutamatergic circuits as a neurosteroid when its concentration is elevated in pregnancy.

Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.

Bone marrow stroma cells (MSCs) have been shown to differentiate into multiple lineages and have great potential for regenerative therapy. We have obtained hMSCs from 6 human adults. They were all positive for CD13, CD44, and CD90 and weakly positive for CD49, while negative for CD45, suggesting that they were different from hematopoetic stem cells. hMSCs were induced to become neuronal cells and maintained as long as three weeks in the serum free medium supplemented with N2. An increase in the amount of mRNA was observed for the NeuroD1, neurofilament M and H, MAP2, neuron-specific enolase, tryptophan hydroxylase, Nurr1, and neuron specific Na⁺ channel genes, and the existence of voltage-gated Na⁺ channels that were sensitive to tetrodotoxin was confirmed electro physiologically. These results suggested that hMSCs differentiated into serotonergic neural cells. However, the expression of genes specific for stroma cells, the Big-h3 and vimentin-genes, was observed equally during the induction process, indicating that the expression pattern was not the completely same as in genuine neural cells. hMSCs cultured with serial passages showed aging phenomena at the cellular level under the various conditions examined in this study. Trials to isolate cellular clones proliferating indefinitely have not succeeded.