Studies Of Retinoic Acid Or Its Derivant-induced Neural Differentiation From Embryonic Stem Cells And Its Application

Embryonic stem cells (ES cells) are self-renewable and show a pluripotent ability to differentiate into cellular derivatives of all three primary germ layers. They are considered as a unique bio-system for disclosing the mechanisms of pluripotency and lineage commitment, and as a useful material for disease modeling, pharmacological screening and cell therapy.Embryonic stem cells can give rise to ectodermal derivatives in culture and can be further induced into neurons and glia. Alltrans retinoic acid (RA) is known to be capable of inducing human or mouse ES cells to differentiate into neural cells within embryoid bodies (EBs) in vitro. However, the route by which RA influences neural commitment is still obscure. The components in serum are complicated. And it is difficult to dissect and manipulate differentiation within EBs because they are multicellular agglomerations of extra-embryonic endoderm and definitive ectodermal, mesodermal and endodermal derivatives. In this study, to simplify the differentiation condition, we took the advantage of a monolayer culture system of neural induction and treated the cultures with variant concentrations of RA without the interference with serum. Experiments with pathway inhibitors demonstrated the mechanism of RA as a neural inducer. We found that in RA-treated cultures, the specific marker for ES cells, i.e., Oct4, was down-regulated more rapidly, and markers for neural cells were up-regulated sooner than the other two control groups. With the stimuli of RA, RA nuclear receptors---retinoic acid receptors (RARs) and retinoid X receptors (RXRs), and RA-metabolizing enzymes (CYP26a1 and Raldh2) showed dynamic changes in expression. We reported that RA-promoted neural differentiation could be blocked by RARα-selective antagonist Ro 41-5253, which indicated that RA signaling was essential for neural specification in this monolayer culture system, and RARαpathway might play a key role in this effect. Furthermore, our data demonstrated that RA had crosstalk with ERK and Wnt pathways in the process of this enhanced neural differentiation. N-all-trans-retinoyl-L-proline (ATRP) is a new compound, synthesized based on the structure of alltrans retinoic acid (RA) and fenretinide. Previous report shows that it has metastasis inhibition activity in hepatocellular carcinoma cells (HCC). However, data on the neural differentiation effects of this new compound on embryonic stem cells were not available. Our results show that higher percentage of neural precursor cells could be gained by using ATRP than by using RA. Upon the addition of ATRP, differentiation was directed rapidly into the neural lineage. During ATRP-induced neural differentiation, genes of retinoic acid nuclear receptors showed dynamic changes. Pharmacological interference with retinoic acid receptor alpha (RARα) signaling suppressed the neural fate choice. Inhibition of FGF signaling could not significantly suppress neural differentiation in ATRP-treated cultures. Both interference with ERK pathway and activation of Wnt pathway could effectively block the ATRP-promoted neural specification. And ERK phosphorylation was enhanced in ATRP-treated cultures at the early stage of differentiation. These data demonstrate that ATRP is an enhancer of neural differentiation in murine embryonic stem cells, and RARαpathway, ERK pathway and Wnt pathway are involved in ATRP-promoted neural specification. However, it is worth noting that how ATRP works with RARαand whether ATRP could transfer into RA during its metabolism need further investigation.Alzheimer's disease (AD) is a neurodegenerative disorder that affects the elderly population and is characterized clinically by progressive loss of memory and decline of multiple cognitive abilities. The major neuropathological features of AD are synaptic and neuronal degeneration, and the presence of amyloid plaques. Current drug treatments mainly aim at alleviating the cholinergic deficits, but unfortunately, these strategies produce only marginal benefits at the risk of adverse side effects. We employed Mo/Hu APPswe PS1dE9 double transgenic mouse as a mouse model of AD and used RA-treated mouse embryonic stem cell-derived neural stem cells (ES-NSCs) as grafts. These ES-NSCs contained nestin-positive cells as the major population, which were able to differentiate into neurons and astrocytes in vitro. Transplantation of ES-NSCs into the lateral ventricle (LV) resulted in the appearance of MAP2- or ChAT-positive neurons derived from the grafted cells in the cerebral cortex, where many Aβdeposits took place. Furthermore, ES-NSCs transplantation improved cognitive function in AD mice assessed by Morris water maze. It might be an effective delivery system for neuronal lineage-committed progenitor cells moving toward the pathological sites of AD via intraventricular transplantation, and these data suggested the potential neuroreplacement therapy of ES-NSCs for AD.