Development of synaptic transmission by cholinergic neurons in culture

We examined the development of cholinergic transmission at synapses formed in culture by retinal neurons derived from the embryonic chick. In our experimental system, striated muscle cells served as postsynaptic targets for cholinergic retinal neurons. Functional retina-muscle synapses could be formed in cultures containing either retinal explants or dissociated retinal cells. Plating a low density of dissociated cells permitted the study of relatively isolated, visually identifiable, cholinergic neurons. We found that, early in the functional maturation of retina-muscle synapses, the release of acetylcholine occurs spontaneously, but cannot be evoked by the putative excitatory transmitter, glutamate. This stage is followed by the emergence of transmitter release that is evocable by glutamate in a receptormediated and calcium-dependent manner. This ability to transmit excitatory information across a synapse is expressed in culture by neurons derived from retinas that are at an early stage of morphogenesis.     

The relationship between chondroprotective and antiinflammatory effects of Withania somnifera root and glucosamine sulphate on human osteoarthritic cartilage in vitro

Using a validated explant model of in vitro cartilage damage, the effects of aqueous extracts of Withania somnifera (Ashwagandha) root and glucosamine sulphate (GlcS) were tested on the levels of nitric oxide (NO) and glycosaminoglycans (GAGs) secreted by knee cartilage from chronic osteoarthritis (OA) patients. W. somnifera extracts significantly decreased NO release by explants from one subset of patients (antiinflammatory response) and significantly increased levels of NO and GAGs released by explants from the second subset ('non-responders'). This is the first study showing direct, statistically significant, antiinflammatory effects of W. somnifera on human OA cartilage. It also confirmed that glucosamine sulphate exhibited statistically significant, antiinflammatory and chondroprotective activities in human OA cartilage. However, these beneficial effects of GlcS were observed in cartilage explants from 50% of patients tested ('responders'). In contrast, glucosamine significantly increased secretion of NO but not GAGs in explants from the second subset of OA patients ('non-responders'). Cartilage explants from the 11 OA patients gave differential responses to both drugs. Patient samples which responded to the antiinflammatory effects of W. somnifera did not always give a similar response to glucosamine, and vice versa. Thus, this in vitro model of human cartilage damage provides qualitative and statistically significant, quantitative pre-clinical data on antiinflammatory and chondroprotective activities of antiarthritic drugs.     

Effects of melanocortin receptor ligands on thyrotropin-releasing hormone release: evidence for the differential roles of melanocortin 3 and 4 receptors

The hypothalamic melanocortin system is important in the central regulation of food intake and body weight. We have previously demonstrated that intracerebroventricular administration of alpha-melanocyte stimulating hormone (alpha-MSH), a nonselective MC3 and MC4 receptor agonist, stimulated plasma thyroid-stimulating hormone, and agouti-related protein (AgRP), an MC3 and MC4 receptor antagonist, suppressed it. In this study, we investigated the effects of MC3 and MC4 receptor (MC3-R and MC4-R) selective agonists and antagonists on the release of thyrotropin-releasing hormone (TRH) from hypothalamic explants in vitro. alpha-MSH stimulated TRH release from the rat hypothalamic explants (alpha-MSH 100 nm 230 +/- 22.9% basal, P < 0.005). In contrast, gamma 2-MSH, a selective MC3-R agonist, suppressed TRH release (gamma 2-MSH 10 microns 76.2 +/- 7.4% basal, P < 0.05). AgRP (83-132), a nonselective MC3/4-R antagonist, induced no change in TRH release whilst JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH 11-22), a selective MC4-R antagonist, suppressed it (JKC-363 10 nm 57.2 +/- 11.5% basal, P < 0.05). Both AgRP (83-132) and JKC-363 blocked alpha-MSH stimulated TRH release but only AgRP (83-132) blocked the inhibitory effect of gamma 2-MSH on TRH release. These data suggest differential roles for the MC3 and MC4 receptors in TRH release; MC3-R agonism inhibiting and MC4-R agonism stimulating TRH release.     

仙茅叶片的组织培养及其细胞学观察

目的通过对仙茅叶片组织培养的研究与细胞学观察,为仙茅的快速繁殖提供技术依据。方法将仙茅幼叶培养在MS培养基,通过设计不同的光照条件,附加不同的激素、水解酪蛋白和活性炭成分,调查出愈率和成苗率。细胞学观察采用石蜡切片法。结果对仙茅叶片的愈伤诱导,黑暗的效果好于光照;在所试验的培养基成分中,适宜的激素配比是2.0mg/L2,4-D或6-BA1.5mg/L 2,4-D2.5mg/L,并且附加300mg/L水解酪蛋白和0.2%活性炭,对于仙茅叶片的离体成苗较好;培养后,愈伤组织主要由叶片的中脉产生,位于中脉上表皮内侧的薄壁细胞首先启动分裂,随后维管束鞘薄壁细胞及其外侧的叶肉细胞也启动分裂,参与愈伤组织的形成。愈伤分化时,芽发生于愈伤组织的表面,根发生于愈伤组织的内部。结论可以通过仙茅幼叶的组织培养进行仙茅的快速繁殖。     

Different requirements for GATA factors in cardiogenesis are mediated by non‐canonical Wnt signaling

GATA factors and Wnt signals are key regulators of vertebrate cardiogenesis, but specific roles for individual GATA factors and how they interact with Wnt signaling remain unknown. We use loss of function and overexpression approaches to elucidate how these molecules regulate early cardiogenesis in Xenopus. In order to minimize indirect effects due to abnormal early embryogenesis, we use pluripotent embryonic tissues as cardiogenic assays. We confirm central roles for GATA4, 5, and 6 in cardiogenesis, but also discover individual and different requirements. We show that GATA4 or 6 regulate both cardiogenic potential and subsequent cardiomyocyte differentiation but that GATA5 is involved in regulating cardiomyocyte differentiation. We also show that Wnt11b signaling can rescue reduced cardiac differentiation resulting from loss of function of GATA4 and 6 but not GATA5. We conclude that Wnt11b mediates the differential requirements for GATA factors during vertebrate cardiogenesis. Developmental Dynamics 240:649–662, 2011. © 2011 Wiley‐Liss, Inc.     

大鼠脊髓前角与盆神经节之间植块联合培养的实验研究

目的:联合培养大鼠脊髓前角与盆神经节( major pelvic ganglia,MPG )植块并初步探讨两者相互作用的可能性?方法:建立大鼠脊髓前角与盆神经节之间植块的联合培养体系,观察神经元的生长变化?结果:神经组织在体外联合培养时均生长良好,脊髓前角植块生长出的运动神经元(spinal motor neurons,SMNs)发出突起与盆神经节的神经元形成明显形态上的接触?结论:联合培养的不同系统的神经元能彼此形成明显接触,初步提示运动神经元与盆神经节神经元之间可能形成功能性的相互作用?     

Fibromuscular proliferates induced in vitro using a trans-filter culture system

A trans-filter co-culture system of vascular smooth muscle cells (SMC) and endothelial cells (EC) is compared with a trans-filter culture of SMC without EC. Explants from the tunica media of rabbit aorta are cultured on one side of a polycarbonate filter with a defined pore size (5 microns). Smooth muscle cells grow out from the media explants and migrate through the filter pores to the other side of the filter, where they proliferate. After 14 days in trans-filter culture, a multilayer resembling "fibromuscular plaques" seen in vivo is formed on the opposite side of the filter. Moreover, the proliferation rate of SMC that have migrated through the filter pores is demonstrated to be similar to that of SMC in intimal lesions induced by balloon catheter injury. In the trans-filter co-culture, a monolayer of endothelial cells (porcine, bovine, or human) is cultivated on one side of the filter. Media explants are placed on the other side. This arrangement mimics the in vivo situation of an arterial vessel wall with endothelium and media separated by a porous lamina. In this co-culture system, the few smooth muscle cells that migrate into the subendothelial space do not form a cell multilayer. A confluent endothelial cell layer is capable of inhibiting the formation of a proliferate in this co-culture system.     

Lectin binding to neurites of goldfish retinal explants

The lectin binding characteristics of goldfish retinal explants were examined by fluoresence microscopy. Neurites grown out from cultured retinal explants were found to bind concanavalin A, wheat germ agglutinin and ricin (agglutinin). The effects of tissue fixation on lectin binding to retinal explant neurites suggest that glycolipids may constitute the predominant ricin binding sites. A reduction in labeling with wheat germ agglutinin following sialidase treatment indicates preferential binding of the lectin to sialic acid residues in the neurite membrane. Neurite morphology was unaltered by brief exposure to concanavalin A or wheat germ agglutinin, while ricin caused a marked deteriaration.     

Culture conditions have an impact on the maturation of traceable,transplantable mouse embryonic stem cell‐derived otic progenitor cells

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1‐green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two‐step short‐term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF‐β inhibitors and insulin‐like growth factor IGF‐1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin‐injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Isolation,Culture and Morphological Assessment of Primary Cell Lines from Human Primary Oral Squamous Cell Carcinoma Using Explant Technique

Background: Due to many uses of cell culture in cell biology, biotechnology, and medical research, this technique has evolved into a widely used and accepted methodology. The isolation of primary cells from primary cancer tissue is a crucial step in cell culture technology since it offers a trustworthy source for studying the biology, morphology, and molecular evaluation of cancer cells, just like in the oral cavity tissue of patients. Therefore, the technique used for the isolation, culture, and evaluation of these cells is crucial. Aim: The aim of the present study is to isolate and culture the cells from human primary Oral Squamous Cell Carcinoma [OSCC] tissue and evaluate them for morphological variations using an explant method. Materials and Methods: The patients with OSCC who were undergoing surgery provided the tissue samples. An explant technique was used to achieve the isolation of cells from tissue samples. Following that, the cells were maintained, subcultured, and stored in accordance with the standard American Type Culture Collection [ATCC] protocol. Routine Hematoxylin & Eosin and crystal violet stains were used. These cells were morphologically studied, and the results were assessed for further studies. Results: We were able to successfully isolate and culture cells from 4 different tissue samples using the explant method. Morphological analysis revealed that one tissue had a significantly distinct presentation of epithelial and stromal cells, whereas the other three tissues had only minor morphological differences predominantly stromal cells. Two tissues were discarded after showing contamination. Conclusion: Tissue culture should be done very meticulously specially when oral cavity tissue is used as it is house for millions of microorganisms. The technique must also be thoroughly followed and adjusted accordingly. Using common, inexpensive stains like Hematoxylin and Eosin and crystal violet, which are of great help for examining the morphology of cells routinely.