Separation of planarian neoblasts based on density gradient centrifugation

For highly purified preparations of neoblasts, density gradient centrifugation in Percoll solutions (Pertoft et al., 1978) was applied to cell suspensions obtained by disintegrating Dugesia polychroa (Schmidt) in culture medium contained in a Dounce homogenizer (tolerance: 50 µm; one animal 12 mm in length per ml). To reduce the high viscosity caused by mucus, 0.00063% (w/v) of dithiothreitol was added during disintegration and purification. Based on previous experiments (Schürmann & Peter, 1988), five media were compared.For prepurification, four washing steps (differential centrifugations at 500 × g for 5 min each) were followed by subsequent filtration through a series of nylon gauzes (40, 30, 20 and 15 µm mesh size) and a final washing step. The resulting cell suspensions were then fractionated by isopycnic centrifugation (500 × g, 45 min) in one continuous (1.018–1.121 g ml^–1) or one of seven different discontinuous Percoll gradients (Schürmann, 1993). The best yield and highest purity of neoblasts in one fraction was obtained with a four step gradient (1.03–1.09 g ml^–1): the neoblasts (purity: 91%) were concentrated in one sharp band at the boundary between the densities 1.05 and 1.07 g ml^–1. The spherical cells (diameter from 10 to 13 µm in vivo) stained as typical neoblasts (Pedersen, 1959).Primary cultures were obtained with all media. The medium developed by Teshirogi and Tohya (1988) and its isotonic modification (Schürmann, 1993) proved best, resulting in 86% of viable cells without signs of differentiation after 17 days of culture at 18 °C, with still 46% being left after 31 days. Earlier reports state that isolated neoblasts only survive for 4 days (Betchaku, 1967) and total planarian cell suspensions only 2–3 weeks (Teshirogi & Tohya, 1988).     

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM)

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [¹²⁵I]asialoceruloplasmin (parenchymal cells) or heat-denatured [¹²⁵I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.     

Positional information and gonadal differentiation in the planarian Dugesia lugubris (Turbellaria)

The planarian Dugesia lugubris is a balanced hermaphrodite, meaning that male genetic factors are in equilibrium with female factors. Differentiation of the gonads is controlled by the region in which they develop. According to the classical theory of germ cell formation, these cells stem from neoblasts that are induced to differentiate by factors specific to the gonadal regions, factors presumably due to gradients formed by neurosecretory activity of the cephalic ganglia and longitudinal nerve cords. A more recently proposed theory holds that germ cells in regenerates originate not from neoblasts but from dedifferentiated cells and that characteristics of the gonadal regions are determined by direct interactions of cells here. Results of our experiments with homo- and autoplastic grafst support the classical theory. Prepharyngeal portions grafted onto posterior body portions retained their ability to maintain or induce development of ovaries. Postpharyngeal portions grafted onto anterior portions produced only testes even though the brain developed normally in these regenerates. Under these experimental conditions, gonad regeneration took longer than it does in normal regeneration (i.e., that in which body regions are not displaced).Translated, from the French, by Marianne Klauser and Seth Tyler.     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

A rapid method for the preparation of the neutrophil fraction of granulocytes from human blood by centrifugation on isotonic Nycodenz gradients

A rapid method for the preparation of neutrophils of the granulocyte fraction of human blood is described. A leucocyte-rich fraction is loaded onto preformed isotonic Nycodenz gradients, formed by mixing a stock isotonic Nycodenz solution with a NaCl diluent solution. After low speed centrifugation a neutrophil fraction of high viability, yield and purity can be isolated from the gradients.     

A mortalin-like gene is crucial for planarian stem cell viability

In adult organisms, stem cells are crucial to homeostasis and regeneration of damaged tissues. In planarians, adult stem cells (neoblasts) are endowed with an extraordinary replicative potential that guarantees unlimited replacement of all differentiated cell types and extraordinary regenerative ability. The molecular mechanisms by which neoblasts combine long-term stability and constant proliferative activity, overcoming the impact of time, remain by far unknown. Here we investigate the role of Djmot, a planarian orthologue that encodes a peculiar member of the HSP70 family, named Mortalin, on the dynamics of stem cells of Dugesia japonica. Planarian stem cells and progenitors constitutively express Djmot. Transient Djmot expression in differentiated tissues is only observed after X-ray irradiation. DjmotRNA interference causes inability to regenerate and death of the animals, as a result of permanent growth arrest of stem cells. These results provide the first evidence that an hsp-related gene is essential for neoblast viability and suggest the possibility that high levels of Djmot serve to keep a p53-like protein signaling under control, thus allowing neoblasts to escape cell death programs. Further studies are needed to unravel the molecular pathways involved in these processes.     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.     

beta-Glucosidase Activity in Corn Roots: Problems in Subcellular Fractionation

Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that β-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the β-glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed.     

Adenylate cyclase in regenerating tissues of the planarian Dugesia lugubris (O. Schmidt)

Adenylate cyclase (AC) was localized ultracytochemically in certain tissues of the regenerating planarian Dugesia lugubris. Studies were carried out from one hour after injury up to the 5th day of regeneration. It was found that the greatest amount of active AC appears during the initial hours of regeneration in the membranes of the muscle cells near the wound, in the epithelial cells surrounding the wound, and in rhabdite-forming cells and neoblasts.     

Djrho2 is involved in regeneration of visual nerves in Dugesia japonica

The freshwater planarian is a powerful animal model for studying regeneration and stem cell activity in vivo.During regeneration,stem ceils (neoblasts in planarian) migrated to the wounding edge to re-build missing parts of the body.However, proteins involved in regulating cell migration during planarian regeneration have not been studied extensively.Here we report two small GTPase genes (Djrho2 and Djrho3) of Dugesia japonica (strain Pek-1).In situ hybridization results indicated that Djrho2 was expressed throughout the body with the exception of the pharynx region while Djrho3 was specifically expressed along the gastro-vaseular system.Djrho2 was largely expressed in neoblasts since its expression was sensitive to X-ray irradiation.In Djrho2-RNAi planarians, smaller anterior blaste-mas were observed in tail fragments during regeneration.Consistently, defective regeneration of visual nerve was detected by immu-nostainning with VC-1 antibody.These results suggested that Djrho2 is required for proper anterior regeneration in planairan.In contrast,no abnormality was observed after RNAi of Djrho3.We compared protein compositions of control and Djrho2-RNAi planarians using an optimized proteomic approach.Twenty-two up-regulated and 26 de-regulated protein spots were observed in the two-dimensional elec-trophoresis gels, and 17 proteins were successfully identified by Mass Spectrometry (MS) analysis.Among them, 6 actin-binding or cy-toskeleton-related proteins were found de-expressed in Djrho2-RNAi animals, suggesting that abnormal cytoskeleton assembling and cell migration were likely reasons of defected regeneration.     

Independence of buoyant cell density and growth rate in Escherichia coli

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.     

Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies

It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.     

The Planarian HOM/HOX Homeobox Genes (Plox) Expressed along the Anteroposterior Axis

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 ( anarian HOM/H homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141–148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.     

Evaluation of a robotic system for the recovery of peripheral blood mononuclear cells

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.     

A rapid method for isolation of the plasma membrane of the myxamoebae and swarm cells of the myxomycete Didymium iridis.

A method for the isolation of the plasma membranes of the acellular slime mould, Didymium iridis in the myxamoebae and swarm cell stages was developed using a modified dextranpolyethylene glycol aqueous two-phase polymer system. It was found to be far superior to the widely accepted technique of density gradient centrifugation concerning this cellular system. Chemical and enzymatic assays performed on the plasma membranes and other cell fractions as well as microscopic examination were used as a basis for positive identification and assessment of purity. Results of the chemical and enzymatic assays indicate that plasma membranes are recovered with high yield and purity using the modified two-phase polymer technique. The method is both rapid and effective and can be performed using low-speed centrifugation.     

Optimization of yield in magnetic cell separations using nickel nanowires of different lengths

Ferromagnetic nanowires are shown to perform both high yield and high purity single-step cell separations on cultures of NIH-3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin-mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5-35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4-fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin-C.     

Five-in-One: Simultaneous isolation of multiple major liver cell types from livers of normal and NASH mice

NASH is a chronic liver disease that affects 3%–6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.     

Distribution of the stem cells (neoblasts) in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

It has been postulated that the high regeneration ability of planarians is supported by totipotent stem cells, called neoblasts. There have been a few reports showing the distribution of neoblasts in planarians. However, the findings were not completely consistent. To determine the distribution of neoblasts, we focused on proliferating cell nuclear antigen (PCNA), which is present in proliferative cells. We cloned and sequenced the cDNA of PCNA from the planarian Dugesia japonica and produced an antiserum recognizing the gene product. X-ray irradiation caused rapid loss of all PCNA-positive cells and loss of the neoblasts (which were morphologically defined by the presence of the chromatoid body), strongly suggesting that all PCNA-positive cells were true neoblasts. Using the antiserum, we were successful in identifying the neoblasts more clearly than any previous work. In addition to their dispersed distribution in the dorsal and ventral mesenchyme, the neoblasts were distributed as clusters along the midline and bilateral lines in the dorsal mesenchyme. We also examined the behavior of the neoblasts after decapitation. Decapitation did not seem to affect the migration of neoblasts far from the wound. We demonstrated here that DjPCNA is a powerful tool for identifying planarian neoblasts.Edited by D.A. Weisblat     

A resource of single-cell gene expression profiles in a planarian Dugesia japonica

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package “Seurat” and GSE223927, researchers can easily access and utilize this dataset.     

建立同时分离培养小鼠肝细胞及肝星状细胞的技术

目的:建立一种简便、经济、高产的同步分离培养肝细胞以及肝星状细胞的方法。方法:在参照国内外方法的基础上加以改良,首先采用肝脏原位胶原酶灌注消化的方法,获得总细胞悬液,经多次低速离心分离肝细胞;再用Nycodenz作为分离介质,通过密度梯度离心法从非实质细胞中得到肝星状细胞。通过台盼蓝染色方法鉴定细胞的活力,用倒置相差显微镜、立体显微镜、CK-18、白蛋白免疫荧光细胞化学染色对培养的肝细胞形态以及功能进行检测。使用Desmin、α-SMA免疫荧光细胞化学对肝星状细胞进行鉴定。结果:成功的在体外同步分离、培养肝细胞及肝星状细胞,肝细胞产率为5-6×107/只小鼠,两只小鼠肝星状细胞产率达1×106个。细胞存活率及纯度均可达90%。肝细胞在培养24h后呈不规则铺路石样形态,此为典型的肝细胞形态,其标志分子CK-18以及白蛋白免疫荧光染色阳性。倒置相差显微镜下可见贴壁后的肝星状细胞呈典型的星形细胞形态,且其标志分子Desmin、α-SMA免疫荧光染色阳性。结论:改良的原位灌注以及分离方法可以同时分离并且培养具有高活性和功能的肝细胞和肝星状细胞。