Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).     

酪氨酸羟化酶与视网膜疾病

酪氨酸羟化酶(tyrosine hydroxylase,TH)是儿茶酚胺(catecholamines,CAs)合成过程中的限速酶,提高或抑制该酶的活性可大幅度影响CAs的合成。近年来对于TH的关注越来越多,大量的研究表明,TH含量及活性的异常改变能通过影响CAs的含量而导致相应生理功能的异常,从而导致疾病。本文将TH的结构功能、分布、表达变化及其与视网膜疾病关系做一综述。     

酪氨酸羟化酶与视网膜疾病

酪氨酸羟化酶（tyrosine hydroxylase,TH）是儿茶酚胺（catech01amines,CAs）合成过程中的限速酶,提高或抑制该酶的活性可大幅度影响CAs的合成。近年来对于TH的关注越来越多,大量的研究表明,TH含量及活性的异常改变能通过影响CAs的含量而导致相应生理功能的异常,从而导致疾病。本文将TH的结构功能、分布、表达变化及其与视网膜疾病关系做一综述。     

ReNCell VM conditioned medium enhances the induction of dental pulp stem cells into dopaminergic like cells

Among the debilitating diseases, neurological related diseases are the most challenging ones to be treated using cell replacement therapies. Recently, dental pulp stem cells (SHED) were found to be most suitable cell choice for neurological related diseases as evidenced with many preclinical studies. To enhance the neurological potential of SHED, we recapitulated one of the pharmacological therapeutic tools in cell replacement treatment, we pre-conditioned dental pulp stem cells (SHED) with culture medium of ReNCell VM, an immortalized neuron progenitor cell, prior to neurogenesis induction and investigated whether this practice enhances their neurogenesis potential especially towards dopaminergic neurons. We hypothesed that the integration of pharmacological practices such as co-administration of various drugs, a wide range of doses and duration as well as pre-conditioning into cell replacement may enhance the efficacy of stem cell therapy. In particular, pre-conditioning is shown to be involved in the protective effect from some membrano-tropic drugs, thereby improving the resistance of cell structures and homing capabilities. We found that cells pre-treated with ReNCell VM conditioned medium displayed bipolar structures with extensive branches resembling putative dopaminergic neurons as compared to non-treated cells. Furthermore, many neuronal related markers such as NES, NR4A2, MSI1, and TH were highly expressed (fold changes > 2; p < 0.05) in pre-treated cells. Similar observations were detected at the protein level. The results demonstrate for the first time that SHED pre-conditioning enhances neurological potential and we suggest that cells should be primed to their respective environment prior to transplantation.     

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co‐culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH‐positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.     

Pluripotent stem cell-derived neural stem cells: From basic research to applications

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.     

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.     

锂对造血干细胞和神经干细胞作用影响的研究进展

锂在现代精神病学中使用超过65年,其构成了双相情感障碍（BD）长期治疗的基础。锂的许多生物学特性已经被证实,包括抗病毒、血液系统和神经系统保护作用。本文系统综述了锂对造血干细胞（HSCs）、神经干细胞（NSCs）以及诱导多能干细胞（iPSCs）作用影响的研究进展及其目前已证实的分子机制。自20世纪70年代以来,锂对保持HSCs和生长因子高水平的作用已被报道。锂可以改善HSCs的归巢能力、形成菌落的能力和自我更新的能力。关于锂对神经发生影响的研究表明,锂可促进海马齿状回的干细胞增殖,并导致施旺氏细胞有丝分裂活性增强。锂被证实与神经保护和神经营养作用相关,具体作用反映在锂可改善突触的可塑性,促进细胞存活,抑制细胞凋亡等。在临床研究中发现,锂离子的治疗可增加大脑灰质的成分,尤其作用在额叶、海马和杏仁核等位置。锂对干细胞的作用涉及多条介质和信号通路,其中最重要的介质和信号通路被认为是糖原合成酶激酶-3（GSK-3）和Wnt/β-catenin通路,另外包括调节cAMP、蛋白激酶B、磷脂酰肌醇3-激酶（pi3k）和肌醇单磷酸酶（IMP）水平的信号通路等也与锂作用有紧密的联系。锂在现阶段被利用于治疗BD和降低痴呆症患病风险的临床实验中,并对神经退行性疾病发挥有益作用。除此之外,为了研究的发病机制和锂离子在其中的作用机制,从BD患者中获得的iPSCs也被广泛应用。     

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.     

Embryonic stem cells in non-human primates: An overview of neural differentiation potential

Non-human primate (NHP) embryonic stem (ES) cells show unlimited proliferative capacities and a great potential to generate multiple cell lineages. These properties make them an ideal resource both for investigating early developmental processes and for assessing their therapeutic potential in numerous models of degenerative diseases. They share the same markers and the same properties with human ES cells, and thus provide an invaluable transitional model that can be used to address the safety issues related to the clinical use of human ES cells. Here, we review the available information on the derivation and the specific features of monkey ES cells. We comment on the capacity of primate ES cells to differentiate into neural lineages and the current protocols to generate self-renewing neural stem cells. We also highlight the signalling pathways involved in the maintenance of these neural cell types. Finally, we discuss the potential of monkey ES cells for neuronal differentiation.     

体外诱导鸡胚胎生殖细胞分化为神经干细胞

本研究探讨体外诱导鸡胚胎生殖细胞(EGCs)分化为神经干细胞(NSCs)的可能性.EGCs经类胚体(EB)阶段,以维生素A酸(RA)等进行诱导,在NSCs选择性培养基中筛培养扩增7 d,观察形态变化;采用RT-PCR法检测nestin基因表达及免疫细胞化学法检测nestin等NSCs特异性标志物,并对其扩增及分化能力进行观察.结果显示:EGCs经初级诱导,NSCs选择性培养基筛选培养7 d后,形成大量神经球样结构,可扩增传代;绝大部分神经球样结构呈nestin抗原阳性,表达nestin基因,且可分化为神经上皮样及少突胶质细胞.研究结果表明:RA等诱导的EGCs,经选择性培养基筛选培养可获得NSCs,有望为眼部神经变性疾病的治疗提供新的技术参考.     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Enhanced neural differentiation of neural stem cells and neurite growth by amniotic epithelial cell co-culture

Amniotic epithelial cells (AECs) were reported to show a neuroprotective effect on neurons, but there was no direct evidence for a functional relationship between neural stem cells (NSCs) and AECs. The aim of this study was to determine whether AECs could stimulate differentiation and expand neurogenesis of NSCs, and whether the roles were due to a diffusible factor or required direct cell-cell contact. AECs were isolated from rat amnion on E14-16 and NSCs were isolated from neocortical tissue. The growth and differentiation of NSCs were compared under different conditions. The results showed that NSCs cultured with FGF-2 proliferated and formed floating neurospheres while those grown in B27 without FGF-2 failed to thrive. Those grown either with AEC conditioned medium or in transwells showed significantly improved survival. Moreover, the neural differentiation and length of neurite were greater in exogenous FGF groups when NSCs were allowed direct contact with AECs. Western blotting, immunocytochemistry and RT-PCR indicated that rat AECs could secrete NT-3 and BDNF. Furthermore, the presence of FGF-2 enhanced the function of AECs. These findings identified that AECs may be regarded as a critical component of NSCs niche and suggested that direct cell-to-cell contact may provide additional and independent support. Such information would circumvent the need for AECs-NSCs co-culture and could potentially facilitate the production of neurons for future clinical applications.     

Adult stem cells in neural repair: Current options,limitations and perspectives

Stem cells represent a promising step for the future of regenerative medicine. As they are able to differentiate into any cell type, tissue or organ, these cells are great candidates for treatments against the worst diseasesthat defy doctors and researchers around the world. Stem cells can be divided into three main groups:(1) embryonic stem cells;(2) fetal stem cells; and(3) adult stem cells. In terms of their capacity for proliferation, stem cells are also classified as totipotent, pluripotent or multipotent. Adult stem cells, also known as somatic cells, are found in various regions of the adult organism, such as bone marrow, skin, eyes, viscera and brain. They can differentiate into unipotent cells of the residing tissue, generally for the purpose of repair. These cells represent an excellent choice in regenerative medicine, every patient can be a donor of adult stem cells to provide a more customized and efficient therapy against various diseases, in other words, they allow the opportunity of autologous transplantation. But in order to start clinical trials and achieve great results, we need to understand how these cells interact with the host tissue, how they can manipulate or be manipulated by the microenvironment where they will be transplanted and for how long they can maintain their multipotent state to provide a full regeneration.