Youxue Liu

Pre-activation of retinoid signaling facilitates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all‐trans‐retinoic acid (ATRA) pre‐induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01–100 μmol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 μmol/L ATRA pre‐induction significantly improved neuronal differentiation efficiency and neural‐cell survival. Compared with MNM alone induced neural‐like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule‐associated protein‐2 (MAP‐2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line‐derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre‐induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRα and RXRγ (and to a lesser extent, RXRβ) were weakly expressed in MSCs. But the expression of RARα and RARγ was readily detectable, whereas RARβ was undetectable. However, at 24 h after ATRA treatment, the expression of RARβ, not RARα or RARγ, increased significantly. We further found the subnuclear redistribution of RARβ in differentiated neurons, suggesting that RARβ may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre‐activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.

Antioxidant vitamin status during pregnancy in relation to cognitive development in the first two years of life

OBJECTIVE To investigate the correlation of the antioxidant vitamins status (vitamins A, E and C) during pregnancy and the intellectual development of early childhood. METHOD A total of 150 paired maternal-neonatal subjects were recruited into the present study. The serum concentrations of antioxidant vitamins (vitamins A, E and C) in maternal blood and cord blood after delivery were determined by high performance liquid chromatography and the intellectual development was evaluated by Gesell Development Schedules (GDS) at two-years-old. RESULT Children with higher cord serum vitamin E level showed higher scores of motor, adaptive domain and average compared to children with lower cord serum vitamin E level (p<0.01 or 0.05), respectively. Cord serum vitamin A level had significant positive correlation with effect on motor DQs (beta=4.227, p<0.05), and vitamin E level in cord blood showed a positive relation with motor DQ and average DQ (beta=0.329 and 0.1875, respectively, p<0.05) in multiple linear regression model. The language and social DQs were influenced by placental vitamin E transport rate (beta=3.1968 and 3.0194, respectively, p<0.05). The placental transport rate of vitamin E also was a protective factor for the prevalence of motor behavior developmental delay [OR: 0.118, 95% confident interval (95% CI), 0.018-0.765, p=0.0251], personal and social behavior developmental delay (OR: 0.052, 95% CI: 0.004-0.610, p=0.0185) and average developmental delay (OR: 0.041, 95% CI: 0.003-0.642, p=0.0229) in logistic multiple regression model. CONCLUSION Data suggested that vitamin A, E status and vitamin E transfer rate at delivery had beneficial influence on childrens cognitive and behavior development quotients.

Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

BackgroundMesenchymal stem cells (MSCs) can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs), MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs) line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T) antigen as an alternative to primary MSCs.MethodsThe retroviral vector SSR#69 expressing simian virus 40 large T (SV40T) antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy.ResultsIn this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP) compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs played same role as primary MSCs to improve learning ability and spatial memory of HIBD rats.ConclusionsIMSCs not only retain their features of primary MSCs but also possess the ability of high proliferation and anti-senescence. IMSCs can definitely be induced to differentiate into neuronal cells in vitro and take the place of primary MSCs for cell transplantation therapy without tumorigenesis in vivo. The stable cell line is particularly useful and valuable as an alternative to MSCs in neuronal differentiation and neuroregeneration associated studies.

Co-assessment of iron, vitamin A and growth status to investigate anemia in preschool children in suburb Chongqing, China

BackgroundAnemia is a widespread public health problem, which is due to many factors, nutritional or non-nutritional. Iron, vitamin A and growth status were assessed to investigate anemia of preschool children in suburb Chongqing, China.MethodsA descriptive, cross-sectional survey was performed on 459 preschool children aged 2 to 7 years randomly chosen from the kindergartens in 6 suburban districts of Chongqing. Weight and height levels, hemoglobin, erythrocyte protoporphyrin, serum retinol, and ferritin concentrations were measured to evaluate the anthropometric and nutritional status.ResultsThe rates of stunt, underweight, overweight, wasting, obesity, anemia, iron deficiency, vitamin A deficiency (VAD), and marginal VAD were 6.3%, 3.9%, 3.7%, 1.5%, 3.1%, 23.5%, 15.0%, 6.3% and 25.9%, respectively. Serum retinol concentration was significantly lower in children with anemia than in those without anemia (P=0.003), and the retinol concentration was associated with hemoglobin (Pearson’s correlation coefficient, r=0.22, P<0.01). Children with VAD had a significantly increased risk for anemia (odds ratio, 2.56; 95% confident interval, 1.15–5.70). In all 108 children with anemia, only 42 were related to VAD and 12 related to iron deficiency, suggesting that almost half of the anemia children cannot be explained solely by iron deficiency or VAD.ConclusionsVitamin A and iron deficiency are still public health problems in some localities of China. Public health interventions in anemia control should be used to eliminate deficiencies of vitamin A, iron, and other micronutrients by deliberate supplementation. Attention must be paid to such deficiencies in high-risk groups, especially in preschool children.

Vitamin A Deficiency Impairs Spatial Learning and Memory: The Mechanism of Abnormal CBP-Dependent Histone Acetylation Regulated by Retinoic Acid Receptor Alpha

Vitamin A (VA) is an essential micronutrient. Numerous studies have confirmed that VA deficiency (VAD) leads to a decline in learning and memory function. Our previous studies have demonstrated that retinoic acid nuclear receptor α (RARα) in the hippocampus plays a crucial role in learning and memory, but the exact mechanism for this process is unclear. Epigenetic modifications, particularly histone acetylation, are involved in nervous system development, learning and memory function, and the pathogenesis of neurodegenerative diseases. Histone acetyltransferases (HATs), such as CREB-binding protein (CBP), E1A-binding protein p300 (p300), and p300/CBP-associated factor (PCAF), are critical for regulating memory function. The current study uses RARα and CBP as examples to study the connections between the RA signaling pathway and histone acetylation modification and to reveal the epigenetic mechanism in VAD-induced learning and memory impairment. This study examined the expression of RARα, HATs, acetylated histone H3/H4, and memory-related genes (Zif268, cFos, FosB), as well as the interaction of RARα and CBP in the hippocampus of 8-week-old rats. Additionally, the changes shown in vivo were further assessed in primary cultured neurons with the inhibition or overexpression of RARα. We found significantly lower levels of histone acetylation in the VAD rats. Furthermore, this downregulation, which impairs learning and memory, is induced by the dysregulation of CBP-dependent histone acetylation that is mediated by RARα. This work provides a solid theoretical foundation and experimental basis for the importance of ensuring sufficient nutritional VA during pregnancy and early life to prevent impairments of learning and memory in adulthood.

Perinatal vitamin A status in relation to neurodevelopmental outcome at two years of age.

UNLABELLED Information about the effect of antioxidant vitamins nutrition during pregnancy on offsprings intellectual development is extremely limited. OBJECTIVE To investigate the correlation of antioxidant vitamins (Vitamin A, E and C) at delivery and the neurodevelopment of early childhood. METHOD A total of 158 paired maternal-neonatal subjects were recruited. The serum concentrations of vitamin A, E and C in maternal and cord blood after delivery were determined and intellectual development was evaluated by Gesell Development Schedule (GDS) at two years old. RESULT After adjusting for potential confounders, vitamin A placental transport ratio (VA-PTR) was positively associated with motor area development quotients (DQ) and average DQ(p<0.01). Cord VA level was positively related with language area and social area DQ (p<0.05). Nevertheless, there was no significant association between cord VE, VC levels, VE PTR or VC PTR and GDS. The adaptive area and average DQ in high cord VA group was higher than those in low VA group (p<0.05). Cord VA level and VA-PTR were positively associated with birth head circumference and birth weight, respectively. CONCLUSION Our data suggested that adequate vitamin A at delivery had beneficial influence on neonatal birth outcomes and childrens neurodevelopment in later childhood.

AP2α transcriptional activity is essential for retinoid-induced neuronal differentiation of mesenchymal stem cells

Pre-activation of the retinoid signaling pathway by all-trans retinoic acid facilitates neuronal differentiation of mesenchymal stem cells. Using protein/DNA based screening assays, we identified activator protein 2α as an important downstream target of all-trans retinoic acid. Although all-trans retinoic acid treatment significantly increased activator protein 2α transcriptional activity, it did not affect its expression. Inhibition of activator protein 2α with dominant-negative mutants reduced ATRA-induced differentiation of mesenchymal stem cells into neurons and reversed its associated functional recovery of memory impairment in the cell-based treatment of a hypoxic-ischemic brain damage rat model. Dominant-negative mutants of activator protein 2α inhibited the expression of neuronal markers which were induced by retinoic acid receptor β activation. All-trans retinoic acid treatment increased phosphorylation of activator protein 2α and resulted in its nuclear translocation. This was blocked by siRNA-mediated knockdown of retinoic acid receptor β. Furthermore, we found that retinoic acid receptor β directly interacted with activator protein 2α. In summary, the regulation of all-trans retinoic acid on activator protein 2α transcriptional activity was mediated by activation of retinoic acid receptor β and subsequent phosphorylation and nuclear translocation of activator protein 2α. Our results strongly suggest that activator protein 2α transcriptional activity is essential for all-trans retinoic acid-induced neuronal differentiation of mesenchymal stem cells.

Effect of biscuits fortified with different doses of vitamin A on indices of vitamin A status, haemoglobin and physical growth levels of pre-school children in Chongqing.

OBJECTIVE To investigate the efficacy of biscuits fortified with different doses of vitamin A on improving vitamin A deficiency (VAD), anaemia and physical growth of pre-school children. DESIGN A randomised double-masked population-based field interventional trial with a positive control group. SETTING Banan district of Chongqing, China. SUBJECTS A total of 580 pre-school children aged 3-6 years were randomly recruited into four groups. Children in groups I and II were given biscuits fortified with vitamin A at 30 % of the recommended daily intake (RDA) and 100 % of the RDA once a day for 9 and 3 months, respectively. Children in group III received biscuits containing 20,000 IU of vitamin A once a week for 3 months. Initially, the children in group IV received a 200,000 IU vitamin A capsule just once. At the beginning and end of the study, blood samples were collected to measure Hb, serum retinol, retinol-binding protein and prealbumin, and weight and height were measured. RESULTS All the fortification types significantly decreased the prevalence of VAD and anaemia in each group (P < 0.05). The effect of 9-month intervention on group I was the most efficient (P < 0.0045). After intervention, the Z-scores of height-for-age, weight-for-age and weight-for-height in all groups increased markedly compared with baseline (P < 0.05), but no significant difference was observed among the groups. CONCLUSIONS Data indicated that consuming vitamin A-fortified biscuits with daily 100 % RDA for 3 months has the same effect on the improvement of VAD, anaemia and physical growth as did the weekly 20,000 IU and single 200,000 IU administration in pre-school children.

Effect of marginal vitamin A deficiency during pregnancy on retinoic acid receptors and N-methyl-d-aspartate receptor expression in the offspring of rats☆

This study examined whether pregnancy-related marginal vitamin A deficiency (MVAD) influences postnatal development of retinoic acid receptors (RARs) and N-methyl-D-aspartate (NMDA) receptor subunit 1 (NR1) in hippocampus of rat pups. Sixteen female rats were randomized equally into control and MVAD groups. Dams and pups were fed with either a normal control diet or one deficient in vitamin A. Eight female pups in each group were killed at 1 day, 2 weeks, 4 weeks and 8 weeks after birth, respectively. Serum retinol levels were monitored. The messenger RNA (mRNA) and protein expressions and subcellular localization of RARα, RARβ and NR1 in postnatal hippocampus were detected. At 1 day, 2 weeks and 8 weeks after birth, serum retinol levels in the MVAD group were significantly lower than those in the control group. Results of Morris water maze test at 7 weeks of age showed that spatial learning and memory in the MVAD group were affected. Vitamin A deficiency resulted in decreased mRNA levels of RARα, RARβ and NR1 (P<.05). The protein level of RARα and NR1 in the MVAD group was lower than that of the control group (P<.05). There was no significant difference in RARβ between the groups (P>.05). A mass of RARα and NR1 colocalized in hippocampal cell cytoplasm on postnatal day 1. Our data suggested that vitamin A deficiency in pregnancy may affect the postnatal expression of RARα and NR1, affecting learning and memory function in the hippocampus and synaptic plasticity of the calcium signaling pathway.

Changes in the expression and subcellular localization of RARα in the rat hippocampus during postnatal development

Retinoic acid receptors (RARs) are reported to mediate the effects of retinoid acid and participate in the maintenance of normal hippocampal function during embryonic and postnatal stages. RARalpha is the only one that has been reported to be continuously expressed among RARs in the CA1-CA3 areas of the hippocampus, at both the mRNA and the protein level. Here, we show the expression and subcellular localization of RARalpha in granule and pyramidal cells in various regions of the hippocampus during postnatal development of rats. We discovered that the expression level of RARalpha in postnatal hippocampal tissue gradually decreased over time with increasing developmental maturity of the nervous system. Moreover, the subcellular localization of RARalpha expression showed a phenomenon of intracellular translocation during the postnatal development period. This new discovery is inconsistent with a traditional viewpoint according to which RARalpha, as a nuclear transcription factor, is mainly expressed inside nucleus. This phenomenon suggests that RARalpha may have different actions during each stage of hippocampal development.