Exosomes derived from uMSCs promote hair regrowth in alopecia areata through accelerating human hair follicular keratinocyte proliferation and migration

Alopecia areata (AA) is a complex genetic disease that results in hair loss due to an autoimmune-mediated attack on the hair follicle. Mesenchymal stem cells (MSCs) have great potential to induce hair regeneration due to their strong secretion ability and multidirectional differentiation. Recent studies have revealed that the therapeutic potential of MSCs comes from their secretion ability, which can produce large amounts of bioactive substances and regulate the key physiological functions of subjects. The secretion products of MSCs, such as vesicles, exosomes, and conditioned media, have significant advantages in preparing of biological products derived from stem cells. Human umbilical cord mesenchymal stem cells (uMSCs) are the best choice for exosome production. uMSCs are obtained from the human umbilical cord. The umbilical cord is easy to obtain, and the efficiency of uMSCs isolation and culture higher than that of obtaining MSCs from bone marrow or adipose tissue. In this study, we investigated the effects of exosomes released from uMSCs in AA mice. In summary, due to easy isolation and cultivation, simple preparation, and convenient storage, it is possible to obtain uMSCs, or uMSCs exosomes for research and clinical treatment.     

Isolation of functional human trophoblast cells and their partial characterization in primary cell culture

Summary Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Isolation of function human trophoblast cells and their partial characterization in primary cell culture.

Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Gonadotropin-induced luminal formation in the seminiferous tubules and efferent ductules in young frogs: light and electron microscopic study

To clarify the mechanism of fluid secretion in the testes at the time of gonadotropin-induced spermiation, young Rana nigromaculata were used. As a morphological index of fluid secretion, luminal formation of the seminiferous tubules, and efferent ductules were observed. The following changes were seen by the administration of hCG or frog pituitary: first, the luminal formation of the seminiferous tubules was seen; next, tubular expansion became evident, and finally, luminal formation and expansion were observed in the efferent ductules. These changes were preceded by the separation of cell contact among Sertoli cells and of cell contact between Sertoli cells and the cells of efferent ductules only in the center and the swelling of Sertoli cells and cells of efferent ductules. With regard to the flow of fluid at the time of spermiation, the present results indicate the possibility that there is a difference in the ability for fluid secretion between Sertoli cells and the ductule cells.     

网纹瓜单细胞固-液双层培养及体细胞无性系建立(简报)

网纹瓜(Cucumis melo Var.reticulatus)是名贵的优质甜瓜,为葫芦科、甜瓜属植物。我国引种后生长良好,产量高,经济效益好,每年从日本购进大量杂交种子。但由于种子不纯,基因型差异较大,植株不整齐,产量不稳定,而且价格昂贵。因此,选育适合我国种植的     

黑胸散白蚁腹腺的形态学与细微构造

对黑胸散白蚁(Reticulitermes chinensis Snyder)腹腺整体装片和切片进行了描述.腹腺分前、后两部分.“腹腺前部”有两类分泌细胞和一个中央腔.两类分泌细胞中,一类是圆形,个体较大;另一类细胞突起很长,具扁平的核.复盖于腹腺前部的体壁上有许多小管和一些感器,表明腹腺前部的分泌细胞产物可能是经体壁上的小管或者先贮存于中央腔中,再经体壁小管逸出体外.“腹腺后部”由大的椭圆形分泌细胞组成.根据腹腺前部紧贴于第Ⅴ腹节表皮层,而腹腺后部是可动的,并且复盖于腹腺后部的体壁上无排出小管,作者认为这些细胞的分泌物可能是释放入血淋巴中.腹腺前部及腹腺后部分泌细胞的分泌物可能有不同的机能,有待于进一步研究.     

Statistical optimization of single-cell production from Taxus cuspidata plant cell aggregates

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200?×?g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.     

黄胫小车蝗受精囊的亚显微结构

利用组织学方法,观察了黄胫小车蝗Oedaleus infernalis 受精囊的显微与亚显微结构。结果表明,黄胫小车蝗受精囊为单个,由高度卷曲的受精囊管和蚕豆状的端囊构成。受精囊壁主要由表皮层、上皮层、基膜和肌肉层构成;上皮层包含上皮细胞、导管细胞和腺细胞。上皮细胞在靠表皮层的边缘有大量的微绒毛,两相邻上皮细胞的细胞膜相互嵌入,并有细微的突起延伸在导管细胞及腺细胞之间,直到基膜,达基膜处的上皮细胞膜折叠,与腺细胞膜的折叠,一起形成迷宫样的指状突起,附着在基膜上。导管细胞有一个较大的核和分泌导管,连接于腺细胞的细胞腔和受精囊腔,将腺细胞中分泌物运输到受精囊腔中。腺细胞具有典型的分泌细胞特征： 含发达内质网、高尔基复合体及不同大小的囊泡。肌肉层位于受精囊最外层,附在基膜上。在受精囊不同部位的结构有差异。在交配前和交配后,受精囊腺细胞的亚显微结构也有差异。     

The fine structure of the ‘accessory salivary glands’ of the tettigoniid, Homorocoryphus nitidulus

A study has been carried out to determine the anatomy and fine structure of these apparently undescribed glands. Two major types of cell are present: secretory and epithelial. The secretory cells contain a much branched extracellular chamber into which secretion is discharged and from which it is carried to the median cuticle-lined duct by fine cuticular ductules.     

Efferent ductules of the fan-throated lizard Sitana ponticeriana Cuvier: light and transmission electron microscopy study

Light microscopy histology of efferent ductules and the ultrastructural organization of their epithelium were studied in the fan‐throated lizard Sitana ponticeriana Cuvier. The ductules of this lizard are extra‐testicular and arise from an extra‐testicular rete testis. A major portion of the ductules is intra‐epididymal and occupies the cephalic end of the epididymis. The ductules differentiate histologically into proximal and distal portions. The epithelium is formed of two major tall columnar cell types, the non‐ciliated and ciliated, and one minor cell type, the basal cells. Dark cells were also identified. The non‐ciliated cells possess microvilli towards the luminal end, tubular coated pits at the bases of the microvilli, coated vesicles in the apical cytoplasm and multivesicular bodies, lysosomes and mitochondria in the supranuclear and perinuclear cytoplasm, which reflects their role in the uptake of the material they are processing. These cells also participate in spermiophagy. The ciliated cells reflect their role in mixing the luminal content and/or its transport to the distal parts of the male tract. The lizard efferent ductules share many features in common with those of mammals and a crocodile and several other features with birds and a turtle. Spermiophagy by the efferent ductules is reported here for the first time in a reptile.     

Production of Mouse Embryoid Bodies with Hepatic Differentiation Potential by Stirred Tank Bioreactor

Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.     

Production of mouse embryoid bodies with hepatic differentiation potential by stirred tank bioreactor

Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.     

Differential Centrifugation in Culture and Differentiation of Rat Neural Stem Cells

(1) The study of neural stem cells (NSC) has attracted much attention in recent years because of their therapeutic potential. However, the problem in culture and differentiation of NSC was how to obtain single cell suspension that preserves the function of NSC, and remove the debris caused by mechanical dissociation. In the present study, we try to find a simple and effective way to address the problem, i.e. differential centrifugation. (2) After a gentle mechanical dissociation using Pasteur pipette, the suspension was first centrifuged at 100 g for 5 min, and then recentrifuged at 400 g for 6 min. Finally, the two deposits were resuspended and seeded into culture flask respectively. The suspension from the second deposit was allowed for further culture and differentiation. Immunofluorescence technique was used to identify neural stem cell, neuron, astrocyte, and oligodendrocyte. (3) After the second differential centrifugation, single cell suspension was obtained with 2–3 cell clusters, and the cells not only grew to form neurospheres, but also differentiated into neurons, astrocytes, and oligodendrocytes. (4) Differential centrifugation is a simple and effective way to obtain single cell suspension, which will help make large-scale production of neurodifferentiated cells more effective.     

Morphology of the salivary glands of Mastotermes darwiniensis Froggatt (Isoptera : Mastotermitidae)

The salivary glands of Mastotermes darwiniensis (Isoptera : Mastotermitidae) workers consist of acini made up of peripherally and centrally placed cells with storage cells. A network of small efferent ductules is associated with each acinus. Intracellular ductules of the central and peripheral cells are filled with protruding microvilli. Central cells contain an abundance of rough endoplasmic reticulum and vacuoles. Continua of rough endoplasmic reticulum and vacuoles are present. Pinocytosis does not appear to be involved in secretion by the central cells. Peripheral cells are compartmental, having electron-lucent vesicle accumulations away from intracellular ductules and the basal plasmalemma invaginations. Peripheral cell morphology is similar to cells which are associated with fluid movement. Storage cells contain a large vacuole and exhibit micropinocytosis. Two axon profile types are present near and on the acini and salivary ducts, one of which appears associated with neuronal end plates.     

Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator.

In order to facilitate the isolation of pure cultures from natural habitats we have developed a method for the isolation of single microbial cell clones from a mixed population, e.g. the flora of the termite gut, with the aid of a modern micromanipulator. The separated single prokaryotic or eukaryotic cells were grown after transfer in culture media or they were used for single cell PCR. The micromanipulator was also applied for the removal of nuclei from protozoa, of which the SSU rDNA was directly amplified.     

Efferent ductules of the boar--a morphological study.

The efferent ductules of the boar were investigated by means of corrosion casts, light microscopy, scanning and transmission electron microscopy. They arise from an extratesticular rete and constitute the major, caudolateral part of the ascending limb of the caput epididymidis. Ductules may be subdivided into three segments: a slightly undulating testicular segment, a highly coiled intermediate segment and a moderately coiled epididymal segment. A decrease in diameter is particularly marked from the intermediate to the epididymal segment. The epithelial transitions from the extratesticular rete to the efferent ductules and from these to the epididymal duct are clearly demarcated. The epithelium of the efferent ducts consists of principal and ciliated cells. Mononuclear leukocytes are found in the basal half. Ultrastructural evidence supports a strong absorptive activity of principal cells. Apical protrusions are not considered to be a proof of apocrine secretion but rather seem to be artifacts. The nature of membrane-bound granules of variable density remains speculative.     

Secretion from rat basophilic leukaemia cells induced by calcium ionophores Effect of pH and metabolic inhibition

Previous experiments on the functional properties of rat basophilic leukaemia cells showed a major anomaly when compared to normal mast cells: though IgE-mediated secretion was dependent on external Ca²⁺ with both types of cells, substantial non-cytotoxic release with ionophore A23187 could be demonstrated with the normal cells but not with the tumour cells. We now show that when the pH of the incubation medium is increased to 8 it is possible to obtain excellent Ca-dependent, non-cytotoxic secretion from tumour basophils with the ionophores A23187 and ionomycin. These results provide further evidence that secretion from the tumour cells occurs via a mechanism similar to that used by normal mast cells and basophils. Experiments with metabolically inhibited tumour cells suggest that their unusual sensitivity to the cytotoxic effects of Ca²⁺ ionophores may be related to their ability to sequester intracellular calcium. Changes in the conditions of cell culture appeared to produce substantial and at least partially reversible changes in responsiveness to IgE-mediated triggering and ionophores.     

颌突外胚间充质干细胞(EMSCs)的体外分离及鉴定

外胚间充质(ectomesenchyme)是一种胚胎发育早期颅面部出现的多能性结构(multipotentstructure),大多数颅面部结构和组织均由其衍生而来,这提示外胚间充质中存在一种干细胞,即外胚间充质干细胞(ectomesenchymalstemcells,EMSCs)。为了分离和鉴定EMSCs,对E125的SD大鼠颌突组织细胞进行了流式细胞学分析,发现其中的外胚间充质细胞表达多种神经谱系和中胚层谱系的标志,包括p75、CD57和nestin等。根据此特点,采用磁细胞分离技术对p75+的颌突外胚间充质细胞进行了分离和克隆培养。克隆分析表明,单个p75+细胞经过10～14d培养,可以形成由两种或两种以上细胞组成的多潜能性克隆(multipotentclone),提示该群外胚间充质细胞具有多潜能性。同时,亚克隆分析表明,多潜能性子克隆中的单个p75+细胞具有再次形成多潜能性克隆的能力,说明这些细胞在体外具有自我更新的能力。这些结果提示,p75+细胞同时具有多潜能性和自我更新能力,因此是外胚间充质干细胞。该干细胞的分离对于口腔颅面部的起源和发育研究无疑具有重要意义。此外,该干细胞的高度可塑性也预示它可以作为一种新的种子细胞,为组织工程皮肤、肌肉、软骨的研究提供新思路。     

Characterization and differentiation of human first trimester placenta trophoblastic cells in culture.

A preparation of highly enriched isolated cytotrophoblasts was obtained from first trimester placenta using dispase incubation of villous tissue at 4 degrees C, followed by a spontaneous cell release at 37 degrees C. After 24 h of culture, 90-95% of the cells were immunostained by anticytokeratin antibody, showing their epithelial characteristic. After 48 h of culture, these cells differentiated into syncytiotrophoblast, as shown by optic and electron microscopic study. The secretion of hCG, and of its free alpha and beta subunits, and the secretion of hPL were studied as a function of cell culture time. While the level of secreted hCG and its free subunits was stable during 72 h of culture, the hPL level was undetectable during the first 48 h of culture, increasing continuously afterwards. Addition of dibutyryl cAMP from the start or after 96 h of cell culture induced an increase of hCG production and of its free subunits and also stimulated the secretion of hPL. This suggests that these cells maintained the capacity to respond to stimuli which increased intracellular cAMP level. Such a cell culture is of interest in further determining the mechanisms of early gestation involved in the differentiation and growth of placental cytotrophoblasts, and in the regulation of their endocrine functions.     

A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90–98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10⁶ cells/T25 flask or 1 × 10⁵ cells/cm²). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.