Generation of chimeric piglets by injection of embryonic germ cells from inbred Wuzhishan miniature pigs into blastocysts.

Background: Human embryonic stem/germ (ES/EG) cell research poses ethical dilemma, it is therefore critical to establish alternative sources of cells for relevant studies. Considering the similarities between the inbred miniature Wuzhishan pigs (WZSP) and humans, ES/EG from these pigs can serve as potential substitutes in human research. In this study, we reported our results that successfully established stable EG cell lines from the WZSP.

Hyperpolarisation-activated cyclic nucleotide channel 4 (HCN4) involvement in Tourette's syndrome autoimmunity.

Objectives: We previously found that antibodies in Tourette's syndrome (TS) patients' sera reacted with a 120 kDa protein from rat brain tissue. Here, we sought to identify this protein and determine if it was involved in TS pathogenesis.

Methods: The 120 kDa protein was identified using immunoprecipitation, Western blotting, and mass spectrometry.

Calcitonin induces podosome disassembly and detachment of osteoclasts by modulating Pyk2 and Src activities.

Osteoclasts (OCs) attach to the extracellular matrix via specialized attachment structures called podosomes, which form a prominent F-actin-rich ring that is thought to correspond to the sealing zone of resorbing OCs. Calcitonin (CT), a 32-amino acid polypeptide, inhibits bone resorption by decreasing motility, inducing retraction, disassembling podosome, and disrupting the actin-ring structure of OCs. However, the detailed mechanisms of how CT induces the disassembly of podosome and disruption of the adhesive structures in OCs are not well characterized.

Effective cryopreservation of human embryonic stem cells by programmed freezing.

Cryopreservation of human embryonic stem cells is an important and unsolved problem. A computer-controlled programmable cooler is used in the preservation of ES cells. Several effects have been experimentally studied, which include the cooling rate, the seeding temperature, the terminative temperature before the sample is plunged into liquid nitrogen.

Cryopreservation of human embryonic stem cells: a protocol by programmed cooling.

Human embryonic stem (ES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the human ES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research.

Improved cryopreservation of human embryonic stem cells with trehalose.

Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose.

Antineural antibody in patients with Tourette's syndrome and their family members.

It has been proposed that antineural antibodies were present in patients with Tourette's syndrome (TS) and other neuropsychiatric disorders. The purpose of our study was to investigate the presence of antineural antibodies in the individuals with Tourette's syndrome and the family members of TS patients. The sera of four TS patients with no current streptococcal infection, their tic-free family members including father, mother and sibling, and a age-matched control group who were tic free were assayed for antineural antibodies directed against rat tissue and neurons in primary cell culture.

[Effective cryopreservation of human embryonic stem cells by programmed freezing].

Cryopreservation of human embryonic stem cells is an important and unsolved problem. A computer-controlled programmable cooler was used in the preservation of ES cells. Several effects have been experimentally studied, which include the cooling rates, the temperature of seeding, the temperatures before the samples being plunged into liquid nitrogen, and the cryoprotective agents.

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells.

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 106 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice.

The culture and establishment of embryonic germ (EG) cell lines from Chinese mini swine.

As a part of a basic research project on Xeno-transplantion, we have been engaged in the derivation of embryonic stem cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 and a 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, marker characterization as well as by in vitro and in vivo differentiation.

[Establishment and differentiation of murine EG cell lines derived from primordial germ cells].

Primordial germ cells (PGC) were isolated from 8.5, 10.5, 12.

[Expression of retinoic acid receptor gamma gene in ES cells and its effect on their differentiation and apoptosis].

We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells.

The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos.

Primordial germ cells (PGCs), as precursors of mammalian germ lineage, have been gaining more attention as a new resource of pluripotent stem cells, which bring a great possibility to study developmental events of germ cell in vitro and at animal level. EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.

Vasculogenesis from embryonic bodies of murine embryonic stem cells transfected by Tgf-beta1 gene.

Mouse embryonic stem (ES) cells transfected with a 1.7 kb cDNA of porcine transforming growth factor type beta1 (TGFbeta1), known as ES-T cells, were found to be able to differentiate in vitro into cystic embryonic bodies (EBs) with outspread tubular structures. Morphological analysis using light, phase-contrast and electron microscopes revealed that in culture, the EBs of ES-T cells initially developed some flat endothelial-like cells which further proliferated and migrated to form thread structures.

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS-1 proteins.

Human gastric cancer MKN-45 cells were transfected with pULB 3238, a plasmid carrying MVMp NS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectants died, while others remained alive, but the growth features of survived cells were changed. For further study on the antineoplastic function of parvoviral NS-1 protein in vivo, transgenic mice carrying NS-1 genes were established by conventional method.

[The mechanism of vasculogenesis: the critical role of transforming growth factor-beta 1 in the formation of vessel-like structures during the differentiation in vitro of murine embryonic stem cells].

When ES-5 cells were transfected with an exogenous porcine TGF-beta 1 gene, one can obtain clones of genetically modified ES cells with over-expression of the transfected gene. We called the genetically modified ES-5 cells as ES-T cells. When ES-T cells were used to study their differentiation in vitro by all trans-retinoic acid (RA), it was soon noticed that embryoid bodies of ES-T cells can exclusively differentiate into endothelial cells and vessel-like structures, but not in their parent ES-5 cells.

[Expression of exogenous porcine transforming growth factor beta-1 gene in ES cells and its effect on their differentiation in vitro].

A TGF-beta 1 gene expression plasmid was constructed by inserting the porcine 1.7 Kb TGF-beta 1 cDNA into BamHI site of retrovirus vector Dol. The plasmid DNA was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in G418 concentration.

[Expression of human multidrug resistance gene (mdr1) cDNA in murine ES cells and in chimeric mice].

Human multidrug resistance gene (mdr1) was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in colchicine concentration (30, 50, 100, and 200 ng/ml respectively). Finally, we obtained 4 clones that could be stably grown in culture medium with colchicine at 200 ng/ml and designated as ES-mdr1 clones A, B, C, and D. Southern blot analysis of DNA from ES-mdr1 A and D cells digested by Hind III and hybridized with mdr1 cDNA 5 A probe was shown in Fig.

[Induced differentiation of mouse embryonic stem cells by retinoic acid and dibutyryl cyclic adenosine monophosphate].

In vitro induced differentiation of mouse embryonic stem cells (ES-5 cells), derived from 5-day 129 mouse blastocyst was studied with retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (dB-cAMP). RA only or RA with dBcAMP together can both induce monolayer ES-5 cells to differentiate into cells of two types: neuron-like cells and fibroblast-like cells. After treated with 10(-6)mol/L RA for 6 days, the differentiated cells were about 80% of all cells, among which most cells were fibroblast-like cells and others were neuron-like cells.

[Immunohistochemical localization of transforming growth factor beta-1 during the early mouse development].

The distribution of transforming growth factor beta-1 (TGF-beta-1) in the early developing mouse embryos between day 1 and day 12 of gestation was examined by immunohistochemical techniques. Polyclonal rabbit antiserum raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta-1 from human platelets was used. The following results were obtained: 1.