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.

Spectrum analysis for assessing red blood cell aggregation using high-frequency ultrasound array transducer

The purpose of this study is to investigate a spectrum analysis technique for detecting and monitoring red blood cell (RBC) aggregation using a high-frequency array transducer. To assess the feasibility of this approach, the backscattered radio-frequency signal from non-aggregated and aggregated RBC samples with two hematocrit levels were acquired by using a 30-MHz linear array transducer and analyzed in frequency domain. Three parameters such as spectral slope, midband fit and Y intercept were extracted in a static condition. Fresh porcine blood was used and degrees of aggregation were changed by diluting plasma concentration. From the experiments, it was demonstrated that the spectral slope related to a size of scatterer progressively declined as the level of aggregation increased; its mean values at hematocrit of 40% were 1.10 and −0.22 dB/MHz for RBCs suspended in isotonic phosphate buffered saline and solution with 70% plasma concentrations, respectively. For the midband fit and Y intercept, the mean values were increased by 9.1 and 46.4 dB, respectively. These results indicated that the spectrum analysis technique is useful for monitoring RBC aggregation and can be potentially developed for assessing aggregation in clinical applications.

Neural Commitment of Embryonic Stem Cells through the Formation of Embryoid Bodies (EBs)

An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4−/4+ protocol.

Screening of proteins interacting with HSPC016 by yeast two-hybridization technique / 中华皮肤科杂志

Objective To screen and identify proteins interacting with hematopoietic stem/progenitor cell differentiation-related gene HSPC016, and to explore the molecular mechanism involved in the regulation by HSPC016 on the aggregative behavior of dermal papilla cells. Methods By using yeast two-hybridization,HSPC016 gene was sub-cloned into pGBKT7 to construct the bait plasmid (named as pGBKT7-HSPC016) in yeast AH109. The cDNA yeast expression library of human dermal papillae cells in yeast Y187 was screened with the bait plasmid and the proteins interacting with HSPC016 were identified. Yeast two-hybridization retransformation experiment was conducted to exclude the false positive clones and verify the interactions, then,the positive clones were sequenced and analyzed by using bioinformatic methods. Results The bait plasmid pGBKT7-HSPC016 was constructed successfully and there was no self-activation in or toxicity against yeast AH 109. Four proteins,including forkhead family of transcription factors (FOXO 1 ), mitogen-activated protein kinase 11 (MAPK 11 ), phosphoinositide-3-kinase (PIK3R3) and liver X receptor were screened and identified. Bioinformatic analysis revealed that these proteins had close relationship with intracellular energy metabolism and translational regulation. Conclusions HSPC016 may regulate the aggregative behavior of DPCs by regulating the levels of intracellular reactive oxygen species (ROS) and interacting with signaling molecules involved in intracellular energy metabolism and translational regulation.

Study on the cell adhesion activity of CEA on tumor cell lines / 中国免疫学杂志

Cell adhesion activity of CEA on tumor cell lines was studied in the experiment.Single cell suspension of CEA positive ceils LoVo and HeLa aggregated respectively in RPMI1640 completemedium at 37℃ to form cell aggregates in different size.The cell aggregation could be specificly inhibited by anti-CEA polyclonal antibodies and purified CEA antigen.Similar results were obtained using cell adhesion assay.The experiment indicated that CEA is one of cell adhesion molecules.