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.

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice.

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys(3)]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.

[The effects of strict dietary salt restriction on blood pressure and proteinuria in chronic glomerulonephritis patients.].

OBJECTIVE: To evaluate the effects of strict dietary salt restriction on blood pressure and proteinuria in chronic glomerulonephritis (CGN) patients. METHODS: From October 2007 to April 2009, 32 CGN inpatients were enrolled. Among them, 15 patients followed a strict dietary salt restriction menu (sodium 100 mmol/d, potassium 50 mmol/d, protein (0.8 - 1.0) gxkg(-1)xd(-1), calorie (105 - 125) kJxkg(-1)xd(-1)) for 7 days, while the other 17 patients were fed freely offered by hospital as controls. 24 h urinary sodium excretion (24h-UNa) was used to monitor the salt intake. No changes of drug therapy were made during the study. Blood pressure was monitored every day. 24-hour urinary protein and serum biochemical parameter were measured before and after the study. RESULTS: There was no significant difference of baseline 24h-UNa between the two groups [(135.1 +/- 50.4) mmol/d vs (137.4 +/- 28.6) mmol/d)]. During the study, the average 24h-UNa of patients with strict dietary intervention was (97.2 +/- 8.6) mmol/d. Both SBP [(117.7 +/- 10.0) mm Hg (1 mm Hg = 0.133 kPa) vs (106.2 +/- 9.9) mm Hg] and DBP [(76.3 +/- 6.1) mm Hg vs (67.5 +/- 5.5) mm Hg] decreased significantly (P < 0.001). Proteinuria decreased significantly too [1.57 (0.3 - 3.0) g/d vs 0.57 (0.16 - 2.72) g/d, P = 0.006]. The reduction of SBP was positively correlated with the reduction of 24h-UNa (r = 0.572, P = 0.026), while the reduction of proteinuria correlated with both the reduction of SBP (r = 0.568, P = 0.027) and 24h-UNa (r = 0.525, P = 0.044). In the control group, only SBP decreased significantly [(122.6 +/- 15.5) mm Hg vs (115.8 +/- 10.4) mm Hg, P = 0.02] without significant changes of DBP and proteinuria. When comparing the subgroups who took ACEI/ARB from both groups, the reduction of proteinuria was more prominent of those from the study group than the control group [-0.4(-0.95 - 0.07) vs 0.07(-0.39 - 0.42), P = 0.014]. CONCLUSION: Strict dietary salt restriction is effective in reducing blood pressure and proteinuria in CGN patients.