Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors.

BACKGROUND: Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson's disease. Several studies have reported the generation of dopaminergic (DA) neurons from mouse and human fibroblasts by ectopic expression of transcription factors, in which genetic manipulation is associated with potential risks. METHODS: The small molecules and protein factors were selected based on their function to directly induce human fetal lung IMR-90 fibroblasts into DA neuron-like cells. Microscopical, immunocytochemical, and RT-qPCR analyses were used to characterize the morphology, phenotype, and gene expression features of the induced cells. The whole-cell patch-clamp recordings were exploited to measure the electrophysiological properties. RESULTS: Human IMR-90 fibroblasts were rapidly converted into DA neuron-like cells after the chemical induction using small molecules and protein factors, with a yield of approximately 95% positive TUJ1-positive cells. The induced DA neuron-like cells were immunopositive for pan-neuronal markers MAP2, NEUN, and Synapsin 1 and DA markers TH, DDC, DAT, and NURR1. The chemical induction process did not involve a neural progenitor/stem cell intermediate stage. The induced neurons could fire single action potentials, which reflected partially the electrophysiological properties of neurons. CONCLUSION: We developed a chemical cocktail of small molecules and protein factors to convert human fibroblasts into DA neuron-like cells without passing through a neural progenitor/stem cell intermediate stage. The induced DA neuron-like cells from human fibroblasts might provide a cellular source for cell-based therapy of Parkinson's disease in the future.

Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules.

BACKGROUND: Motor neuron degeneration or loss in the spinal cord is the characteristic phenotype of motor neuron diseases or spinal cord injuries. Being proliferative and located near neurons, astrocytes are considered ideal cell sources for regenerating neurons. METHODS: We selected and tested different combinations of the small molecules for inducing the conversion of human and mouse astrocytes into neurons. Microscopic imaging and immunocytochemistry analyses were used to characterize the morphology and phenotype of the induced neurons while RT-qPCR was utilized to analyze changes in gene expression. In addition, whole-cell patch-clamp recordings were measured to examine the electrophysiological properties of induced neurons. RESULTS: The results showed that human astrocytes could be rapidly and efficiently converted into motor neuron-like cells by treatment with defined small molecules, with a yield of over 85% motor neuron-like cells attained. The induced motor neuron-like cells expressed the pan-neuronal markers TUJ1, MAP2, NeuN, and Synapsin 1 and motor neuron markers HB9, ISL1, CHAT, and VAChT. During the conversion process, the cells did not pass through a proliferative neural progenitor cell intermediate. The induced motor neurons were functional, showing the electrophysiological properties of neurons. The same chemical cocktail could induce spinal cord astrocytes from an amyotrophic lateral sclerosis mouse model carrying a SOD1 mutation to become motor neuron-like cells that exhibited a decrease in cell survival and an increase in oxidative stress compared to that observed in wild-type MNs derived from healthy mice. Moreover, the chemical induction reduced oxidative stress in the mutant astrocytes. CONCLUSION: The results of the present study demonstrated the feasibility of chemically converting human and mouse astrocytes into motor neuron-like cells that are useful for neurodegenerative disease modeling and regenerative medicine.

Investigation of the usefulness of zaleplon at two doses to induce afternoon-sleep under noise interference and its effects on psychomotor performance and vestibular function.

BACKGROUND: Military operation personnel often suffer from sleep difficulty because of their work requirements. In this study, we investigated the efficacy of zaleplon at two doses to induce afternoon-sleep under noise interference and its effects on psychomotor performance and vestibular function; we subsequently established the optimal dosage regimen for military operation personnel. METHODS: Twenty-two healthy young male volunteers were recruited for the study. Eight subjects took 10 mg or 15 mg of zaleplon and placebo alternately and then were exposed to noise. Changes in polysomnography (PSG) indices, including sleep latency (SL), sleep efficiency (SE) and sleep structure, were recorded after drug administration. After awakening, the volunteers' subjective judgments of sleep quality and sleepiness were measured. Eight volunteers underwent 3 psychomotor performance tests at a one-week interval, and the psychomotor performance tests were conducted before and after taking zaleplon and placebo. Six volunteers participated in the vestibular function test session, and parameters, including optokinetic nystagmus (OKN), vestibular ocular reflex (VOR), visual-vestibular ocular reflex (VVOR) and vestibular ocular reflex fixation suppression (VOR-Fix), were detected by the same experimental design as described above. The data of sleep observations were subjected to one-way variance analysis. RESULTS: Compared with the placebo group, SL was shortened significantly, and the scores of subjective sleep quality and sleep depth were clearly increased in the zaleplon 10 mg group (P < 0.05). Moreover, the SE and the percent of REM (rapid eye movement) sleep were increased remarkably in the zaleplon 15 mg group (P < 0.01). Furthermore, the SE, percent of REM sleep and scores of subjective sleep depth in the zaleplon 15 mg group were significantly higher than in the zaleplon 10 mg group (P < 0.05). The psychomotor performance did not change significantly after ingestion of 10 mg or 15 mg of zaleplon, whereas the OKN and VOR gains were lower in the two dose groups of zaleplon (P < 0.05) and restored to normal 3 h after drug ingestion. CONCLUSION: Zaleplon is an ideal hypnotic for military personnel, and its hypnotic efficiency is dose-related under noise interference; a 15 mg dose of zaleplon could provide significantly better sleep than a 10 mg dose of zaleplon.

Protective effect of Tianqi Hangli Recipe () extract on high sustained positive acceleration stress-induced myocardial mitochondrial injury in rats.

OBJECTIVE: To explore the protective effect of Tianqi Hangli Recipe () extract (THRE) on high sustained positive acceleration (+Gz) stress-induced myocardial mitochondrial injury in rats. METHODS: Seventy-two male SD rats were randomly assigned to various groups with 12 rats per group: blank control group, stress control group, high +Gz stress group, low-dose THRE group (0.75 g/kg), medium-dose THRE group (1.5 g/kg) and high dose THRE group (3.0 g/kg). Each rat was fifi rstly fed with 20 mL/kg menstruum once a day for 14 days. The rats were then exposed to high +Gz at the 15th day. Myocardial mitochondrial structure, respiratory function, antioxidant capacity and ATPases activities were examined for the comparison after the high +Gz exposure. RESULTS: The rats treated with high +Gz stress showed signififi cant pathological changes: the myocardial mitochondria were swelled, degenerated and decreased, and mitochondrial cristae were broken or disappeared. State 3 respiration and the respiratory control ratio (RCR) were both significantly lower, and state 4 respiration was higher as compared with the blank control group and stress control groups (P <0.01, P <0.05). In addition, the activities of its antioxidant enzymes [superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)] and Na+-K+-ATPase were also decreased (P <0.01 or P <0.05), but the formation of malondialdehyde (MDA) was increased (P <0.01). However, THRE preconditioning could attenuate mitochondrial structural damages and reverse the high +Gz stress-caused changes of parameters about respiration, antioxidant enzymes and ATPases, most of which had no significant difference between the high-dose THRE group and the stress control group. CONCLUSION: THRE can protect high sustained +Gz stress-induced myocardial mitochondrial injury in rats as was shown to ameliorate respiratory function and increase activities of antioxidant enzymes and ATPases.