Techniques of fragile renal organoids transplantation in mice

Purpose: This study aimed to develop a microsurgical technique to transplant extremely fragile renal organoids in vivo, created by in-vitro reaggregation of metanephros from fetal mice. These organoids in reaggregation and development were examined histologically after transplantation under the renal capsule. Methods: Initially, metanephros from fetal mice were enzymatically treated to form single cells, and spheroids were generated in vitro. Under a microscope, the renal capsule was detached to avoid bleeding, and the outer cylinder of the indwelling needle was inserted to detach the renal parenchyma from the renal capsule using water pressure. The reaggregated spheroid was aspirated from the culture plate using a syringe with an indwelling needle outer cylinder and carefully extruded under the capsule. Pathological analysis was performed to evaluate changes in reaggregated spheroids over time and the effects of co-culture of spinal cord and subcapsular implantation on maturation. Results: In vitro, the formation of luminal structures became clearer on day 5. These fragile organoids were successfully implanted without tissue crapes under the renal capsule and formed glomerular. The effect of spinal cord co-transplant was not obvious histrionically. Conclusions: A simple and easy method to transplant fragile spheroids and renal under the renal capsule without damage was developed.

Prevention of rocuronium induced mast cell activation with prophylactic oleuropein rich diet in anesthetized rabbits

Abstract Purpose: The effect of a prophylactic oleuropein-rich diet before anesthesia accompanied by the widely-used steroid-based neuromuscular drug rocuronium on mast cell activation was investigated in the study. Methods: 14 rabbits used in the study. The rabbits in the oleuropein group were given oleuropein-rich extract added to the animals' water at doses of 20 mg/kg oleuropein for 15 days orally. After 15 days, all rabbits in the two groups were given general anesthesia with rocuronium of 1 mg/kg. After 1 day, animals were sacrificed and the liver tissue sections stained with H&E, toluidine blue and tryptase for immunohistochemical study. Results: There was no statistically significant difference between ALT, AST and albumin averages of the oleuropein and control groups (p> 0.05). The tryptase average of the control group was higher than the tryptase average of the oleuropein group and this difference was statistically significant (p=0.003). The T. blue average in the oleuropein group was higher than the control group. However, there was no statistically significant difference between groups (p=0.482). Conclusions: Rocuronium adverse effects, like hypersensitivity and anaphylaxis, may limit routine use of this substance. The use of oleuropein reduced the number of inflammatory cells and prevented degranulation.

Knockout of p16INK4a promotes aggregative growth of dermal papilla cells

Summary Objective: Dermal papilla cells (DPCs) are located in the hair follicles and play an important role in hair growth. These cells have the ability to induce hair follicle formation when they display aggregative behavior. DPCs derived from the androgenetic alopecia (AGA) area undergo premature senescence in vitro, associated with p16INK4a expression. The aim of the current study was to investigate the expression of p16INK4a in aggregative and non-aggregative DPCs and the effect of p16INK4a down-regulation in these cells by adenovirus-mediated RNA interference (RNAi). Method: DPCs were isolated and cultured from healthy human scalp. p16INK4a gene and protein were detected in aggregative and non-aggregative cells. Expression of p16INK4a in DPCs was silenced by infection with rAd5-CDKN1A-1p2shRNA. Cell fate was monitored after infection. The growth of cells was measured by MTT assay. Cell cycle was evaluated by flow cytometry (FCM). Results: DPCs were isolated by digestion and showed aggregative behavior for six passages. The expression of p16INK4a showed a clear upward trend in non-aggregative cells when compared with aggregative group. p16INK4a expression was silenced by rAd5-CDKN1A-1p2shRNA (p<0.05). The p16INK4a-silenced cells grew more rapidly and exhibited a trend towards aggregative growth. There was an increase in the proportion of cells in G1 phase, while those in S phase were reduced after p16INK4a gene silencing (p<0.05). Conclusion: Our results suggest that p16INK4a plays an important role in the premature senescence and aggregative behavior of DPCs. These observations can lead to novel therapeutic strategies for treatment of AGA.

Effect of shift rotation culture on formation and activity of encapsulated hepatocytes aggregates / 浙江大学学报·医学版

To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates.AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC).On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all<0.01).Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.

Microarray analysis of the in vitro granulomatous response to Mycobacterium tuberculosis H37Ra / Análisis con Micromatrices de la respuesta granulomatosa in vitro a Mycobacterium tuberculosis H37Ra

Background: The hallmark of tuberculosis is the granuloma, an organized cellular accumulation playing a key role in host defense against Mycobacterium tuberculosis. These structures sequester and contain mycobacterial cells preventing active disease, while long term maintenance of granulomas leads to latent disease. Clear understanding on mechanisms involved in granuloma formation and maintenance is lacking. Objective: To monitor granuloma formation and to determine gene expression profiles induced during the granulomatous response to M. tuberculosis (H37Ra). Methods: We used a previously characterized in vitro human model. Cellular aggregation was followed daily with microscopy and Wright staining for 5 days. Granulomas were collected at 24h, RNA extracted and hybridized to Affymetrix human microarrays. Results: Daily microscopic examination revealed gradual formation of granulomas in response to mycobacterial infection. Granulomatous structures persisted for 96 h, and then began to disappear. Conclusions: Microarray analysis identified genes in the innate immune response and antigen presentation pathways activated during the in vitro granulomatous response to live mycobacterial cells, revealing very early changes in gene expression of the human granulomatous response.

Antecedentes: La marca histológica de la tuberculosis es el granuloma, una acumulación celular organizada que cumple funciones claves en la defensa del hospedero contra Mycobacterium tuberculosis. Estas estructuras secuestran y confinan a las micobacterias previniendo el desarrollo de enfermedad activa; el mantenimiento a largo plazo de los granulomas conlleva al establecimiento de latencia. Un mejor entendimiento de los mecanismos involucrados en la formación y mantenimiento del granuloma es necesario. Objetivo: Monitorear la formación del granuloma y determinar los patrones de expresión génica inducidos durante la respuesta granulomatosa a M. tuberculosis (H37Ra). Métodos: En este estudio se empleó un modelo in vitro humano previamente caracterizado. La agregación celular fue examinada diariamente mediante microscopia óptica y tinción de Wright por 5 días. Para analizar la expresión génica, los granulomas fueron colectados a las 24 h, se extrajo el RNA sometiéndolo a hibridación a micromatrices de Affymetrix. Resultados: Se observó la formación gradual de granulomas en respuesta a la infección. Los granulomas persistieron por 96 h, y luego se desvanecieron. Conclusiones: Se identificaron genes de la respuesta inmune innata y vías de presentación antigénica activadas durante la respuesta granulomatosa in vitro a células micobacteriales vivas, lo cual reveló alteraciones tempranas de la expresión génica en el inicio de la respuesta granulomatosa humana.

Icariin promotes self-renewal of neural stem cells: an involvement of extracellular regulated kinase signaling pathway / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs).</p><p><b>METHODS</b>NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.</p><p><b>RESULTS</b>ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs.</p><p><b>CONCLUSION</b>ICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.</p>

Evaluation of stress tolerance and fermentative behavior of indigenous Saccharomyces cerevisiae

Sixty six indigenous Saccharomyces cerevisiae strains were evaluated in stressful conditions (temperature, osmolarity, sulphite and ethanol tolerance) and also ability to flocculate. Eighteen strains showed tolerant characteristics to these stressful conditions, growing at 42 ºC, in 0.04% sulphite, 1 mol L-1 NaCl and 12% ethanol. No flocculent characteristics were observed. These strains were evaluated according to their fermentative performance in sugar cane juice. The conversion factors of substrates into ethanol (Yp/s), glycerol (Yg/s) and acetic acid (Yac/s), were calculated. The highest values of Yp/s in sugar cane juice fermentation were obtained by four strains, one isolated from fruit (0.46) and the others from sugar cane (0.45, 0.44 and 0.43). These values were higher than the value obtained using traditional yeast (0.38) currently employed in the Brazilian bioethanol industry. The parameters Yg/s and Yac/s were low for all strains. The UFLA FW221 presented the higher values for parameter related to bioethanol production. Thus, it was tested in co-culture with Lactobacillus fermentum. Besides this, a 20-L vessel for five consecutive batches of fermentation was performed. This strain was genetically stable and remained viable during all batches, producing high amounts of ethanol. The UFLA FW221 isolated from fruit was suitable to produce bioethanol in sugar cane juice. Therefore, the study of the biodiversity of yeasts from different environmental can reveal strains with desired characteristics to industrial applications.

Neovascularization in the carotid atherosclerotic plaque is not associated with inflammatory cell aggregation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Monocytes and macrophages in atherosclerotic plaque lead to plaque instability. The aim of the study was to determine if plaque neovascularization led to inflammation.</p><p><b>METHODS</b>Patients were consecutively enrolled if their carotid intimal media thickness was > 2 mm, as revealed by duplex ultrasound. The patients then underwent dynamic contrast enhanced magnetic resonance imaging (DCE MRI) and fluorine-18 fluorodeoxyglucose ((18)F-FDG) positron emission tomography combined with computed tomography (PET CT). A target to background ratio (TBR) of ≥ 1.25 or < 1.25 served as the cutoff point for the presence and absence of inflammation, respectively.</p><p><b>RESULTS</b>Twenty-six patients underwent bilateral carotid DCE MRI and 24 patients also underwent PET CT. One hundred and fifty-five plaques were evaluated by both DCE MRI and PET CT. There was no significant difference in plaque morphology between the TBR ≥ 1.25 (n = 61) and TBR < 1.25 (n = 94) groups. No significant differences were found in plasma volume and transfer constant between the TBR ≥ 1.25 and TBR < 1.25 groups.</p><p><b>CONCLUSION</b>Our study did not find a significant correlation between plaque neovascularization and the aggregation of inflammatory cells.</p>

Heat shock cognate 71 (HSC71) regulates cellular antiviral response by impairing formation of VISA aggregates

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.

Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 µm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.

Effect of Liangxuehuayu Recipe on hemorheology in rats with blood stasis syndrome

OBJECTIVE@#To investigate the effects of Liangxuehuayu Recipe on hemorheology in rats with blood stasis syndrome induced by mutifactor stimuli.@*METHODS@#SD rats were divided into control, model, Liangxuehuayu Recipe (high, middle and low dose, 18, 9, 4.5 g/kg accordingly). Except the control group, blood stasis model was established in the rest groups. The hemorheological parameters were measured and compared.@*RESULTS@#Blood viscosity at high, moderate and low level in rats with blood stasis significantly increased (P<0.05), but blood viscosity at high level and plasma viscosity was significantly decreased in rats induced by some stimuli after Liangxuehuayu Recipe were intra-gastrically administered for 1 weeks (P<0.01, P<0.05).@*CONCLUSIONS@#Liangxuehuayu Recipe is effective in improving hemorheology, and has important application value in the prevention of occurrence and development of ischemic stroke.

CCN2/CTGF silencing blocks cell aggregation in embryonal carcinoma P19 cell

Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.

The Epithelial-Mesenchymal Transition During Tooth Root Development

Hertwig's epithelial root sheath (HERS) consists of bilayered cells derived from the inner and outer dental epithelia and plays important roles in tooth root formation as well as in the maintenance and regeneration of periodontal tissues. With regards to the fate of HERS, and although previous reports have suggested that this entails the formation of epithelial rests of Malassez, apoptosis or an epithelialmesenchymal transformation (EMT), it is unclear what changes occur in the epithelial cells in this structure. This study examined whether HERS cells undergo EMT using a keratin-14 (K14) cre:ROSA 26 transgenic reporter mouse. The K14 transgene is expressed by many epithelial tissues, including the oral epithelium and the enamel organ. A distinct K14 expression pattern was found in the continuous HERS bi-layer and the epithelial diaphragm were visualized by detecting the beta-galactosidase (lacZ) activity in 1 week postnatal mice. The 2 and 4 week old mice showed a fragmented HERS with cell aggregation along the root surface. However, some of the lacZ-positive dissociated cells along the root surface were not positive for pan-cytokeratin. These results suggest that the K14 transgene is a valuable marker of HERS. In addition, the current data suggest that some of the HERS cells may lose their epithelial properties after fragmentation and subsequently undergo EMT.

A Case of Clear Cell Eccrine Porocarcinoma

Eccrine porocarcinoma (EP) is a rare malignant tumor arising from the intraepidermal eccrine duct. The tumor cells frequently contain glycogen, but prominent clear cell changes in EP are rarely reported. A 78-year-old woman presented with a slightly pruritic, erythematous, verrucous plaque on her left thigh. Histopathological examination revealed intraepidermal tumor cell nests composed of small basaloid cells and duct-like structures lined by periodic acid-Schiff (PAS)-positive cuticles. Besides the typical findings of EP, clear cell changes were predominantly observed in the tumor cell aggregations. Herein we report a case of the clear cell variant of EP rarely reported in previous literature.

The hyperexpressions of putative stem cells in the eutopic endometrium of patients with advanced endometriosis / 대한산부인과학회지

OBJECTIVE: Recently it has been proposed that stem cells may be associated with the pathogenesis of endometriosis. The purposes of this study are to investigate whether the eutopic endometrial cells of women with or without endometriosis show the characteristics of stem cells in vitro and have a difference of the expressions of the undifferentiated stem cell markers as OCT-4 and CXCR4. METHODS: A total of 6 women with advanced endometriosis and a total of 10 women without endometriosis, adenomyosis or leiomyoma were included in this study. The eutopic endometrial cells, which were obtained from the menstrual blood at menstrual cycle day 2 to 4, were cultured in vitro for approximately 2 weeks, subsequently the putative very small stem cells were separated by Percoll density gradient method and were cultured. The expressions of OCT-4 and CXCR4 were analyzed by real time RT-PCR. RESULTS: The eutopic endometrial cells of the group of endometriosis compared with the control group showed the different morphological characteristics in vitro; more commonly heterogeneous supportive cells, very small round cells less than 3 micrometer and 5~15 micrometer sized hyperchromatic round cells. After the separation of very small round cells by Percoll density gradient method, these cells showed the several characteristics of stem cells; self-renewal, asymmetric cell division, colony formation and embryoid body-like formation. Also These cells showed the similar characteristics of very small embryonic-like stem cells; the mobile cells smaller than erythrocyte, the cell migration or adhesion to supportive cells, the sphere formation by cell aggregation and the formation of new differentiated cell by cell fusion. The expressions of OCT-4 and CXCR4 in the group of endometriosis are respectively 5.66 times and 17.69 times as high as the control group (P<0.05). CONCLUSION: The very small round cells less than 3 micrometer and 5~15 micrometer sized hyperchromatic round cells, which showed the several characteristics of stem cells in vitro, were more common in eutopic endometrial cells of patients with endometriosis and the expressions of OCT-4 and CXCR4 were significantly higher. This study suggests that stem cells might play a key role in the pathogenesis of endometriosis and OCT-4 and CXCR4 might be used as a tool for diagnosis or follow-up.

EFFECTS OF HYDROQUINONE ON NEOPLASTIC TRANSFORMATION OF HUMAN EPITHELIAL CELLS IN CULTURE

[Overexpression of human proislet amyloid polypeptide in CHO cells and characterization of its aggregation properties: a cell culture model of protein aggregation in mammalian cells]

To study the mechanisms involved in amyloid formation processes, we made a mammalian cell culture model of amylin aggregation and characterized its properties. Amyloid fibrils were extracted from CHO cells and binding affinity to Thioflavin T and Congo red was investigated. Then the apple-green birefringence of extracted fibrils was detected under polarized light to confirm the presence of aggregated protein in the extracts. To better investigate amyloid formation in CHO cells, we decided to overexpress an amyloidogenic protein in these cells. To do so, we amplified ProIAPP gene and subcloned it in EGFP-N1 expression vector. Then, CHO cells were transfected with EGFP-N1-ProIAPP and EGFP-N1 as a control. The phenotypes of around 100 transfected cells were characterized for several days after the transfection, using fluorescence microscopy. Additionally, the presence of amyloid structure in these cells was detected by Congo red staining under exposure to polarized light. Cell viability assay was performed using Trypan blue staining. Low level natural amyloid was detected in CHO cells. In addition, the ProIAPP-EGFP transfected cells exhibited aggregated phenotype in which the cells with round morphology had far more aggregates than oval ones. We found amyloid fibrils in CHO cells at low level. Overexpression of amylin protein in CHO cells caused aggregation phenotypes. These cells can be used as a model of cell culture for studying protein aggregation. Amyloidogenic properties of this protein could immensely help us to study the mechanisms that are involved in amyloid formation in mammalians including humans

Acrylamide-induced neoplastic transformation of human epithelial cells in culture

PURPOSE Acrylamide is present in significant quantities in a wide range of commonly consumed human foods. Carcinogenic risk of acrylamide through the consumption of food is a great public concern and in controversy, but it is not properly addressed due to the lack of evidence in humans. While a plenty of data is available on the carcinogenicity in animal models, the studies in humans are limited. Thus, the present study attempted to examine the carcinogenic potentials of acrylamide on the human epithelial cell, which is the target cell origin of the most cancers. MATERIAL AND METHOD & RESULT 1. Acrylamide was not cytotoxic up to 100 MICRO M as measured by MTT and LDH assays, indicating a relatively low toxicity of this substance in human epithelial cells. 2. The parameters of neoplastic cellular transformation such as cell saturation density, soft-agar colony formation and cell aggregation were analyzed to examine the carcinogenic potential of acrylamide. 3. The neoplastic transformation was further increased with the co-treatment of TPA 4. Antioxidants blocked the generation of Reactive Oxygen Species(ROS) and the GSH depleting agent dramatically increased the ROS production. 5. mRNA levels of fibronectin following acrylamide exposure was increased in a dose-dependent manner, indicating a possible biomarker of acrylamide-induced cellular transformation. CONCLUSION The present study will provide a valuable basis to compare the interspecies differences in response to carcinogenic potentials of acrylamide. The data on the interspecies differences are essential element in human risk assessment. Thus, our results obtained from the human epithelial cells will contribute to improving the risk assessment of human neoplasm including oral cancer.

La agregación celular en la producción de metabolitos secundarios en cultivos vegetales in vitro / Celular aggregation in secondary metabolite production in in vitro plant cell cultures

El cultivo de células vegetales es una alternativa biotecnológica para la producción de metabolitos secundarios. Sin embargo, la productividad de los sistemas in vitro es menor a la obtenida de plantas. En esta revisión se ilustra la diferenciación y compartamentalización celular como eventos necesarios para la síntesis de metabolitos secundarios en las plantas. Se discute la inducción de la agregación celular en los cultivos in vitro como una de las estrategias para favorecer la acumulación de estos compuestos químicos. Este efecto positivo podría ser explicado como consecuencia de la formación de estructuras morfogénicas y/o por una condición de estrés por limitaciones de oxígeno al interior de los agregados. Finalmente, se muestra que la combinación de la agregación con otras estrategias tales como la selección de líneas celulares, la elicitación y la adición de precursores constituye una alternativa para desarrollar bioprocesos a partir de células vegetales in vitro para la producción de compuestos químicos de alto valor agregado.