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Research Containing: Mice

Shear stress induces preimplantation embryo death that is delayed by the zona pellucida and associated with stress-activated protein kinase-mediated apoptosis

by cfynanon 9 June 2015in Biology & Biotechnology No comment

In this study, we discovered that embryos sense shear stress and sought to characterize the kinetics and the enzymatic mechanisms underlying induction of embryonic lethality by shear stress. Using a rotating wall vessel programmed to produce 1.2 dynes/cm2 shear stress, it was found that shear stress caused lethality within 12 h for E3.5 blastocysts. Embryos developed an approximate 100% increase in mitogen-activated protein kinase 8/9 (formerly known as stress-activated protein kinase/junC kinase 1/2) phosphorylation by 6 h of shear stress that further increased to approximately 350% by 12 h. Terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis was at baseline levels at 6 h and increased to approximately 500% of baseline at 12 h, when irreversible commitment to death occurred. A mitogen-activated protein kinase 8/9 phosphorylation inhibitor, D-JNKI1, was able to inhibit over 50% of the apoptosis, suggesting a causal role for mitogen-activated protein kinase 8/9 phosphorylation in the shear stress-induced lethality. The E2.5 (compacted eight-cell/early morula stage) embryo was more sensitive to shear stress than the E3.5 (early blastocyst stage) embryo. Additionally, zona pellucida removal significantly accelerated shear stress-induced lethality while having no lethal effect on embryos in the static control. In conclusion, preimplantation embryos sense shear stress, chronic shear stress is lethal, and the zona pellucida lessens the lethal and sublethal effects of shear stress. Embryos in vivo would not experience as high a sustained velocity or shear stress as induced experimentally here. Lower shear stresses might induce sufficient mitogen-activated protein kinase 8/9 phosphorylation that would slow growth or cause premature differentiation if the zona pellucida were not intact.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/16571875

Microgravity potentiates stem cell proliferation while sustaining the capability of differentiation

by cfynanon 9 June 2015in Biology & Biotechnology No comment

A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(3) G. Here we report that human mesenchymal stem cells (hMSCs) cultured in a 3D-clinostat (group CL) showed marked proliferation (13-fold in a week) compared with cells cultured under normal conditions of 1 G (group C) (4-fold in a week). Flow cytometry revealed a 6-fold increase in the number of hMSCs double-positive for CD44/CD29 or CD90/CD29 in group CL after 7 days in culture, compared with group C. Telomere length remained the same in cells from both groups during culturing. Group C cells showed increasing expression levels of type II collagen and aggrecan over the culture period, whereas group CL cells showed a decrease to undetectable levels. Pellets of hMSCs from each group were explanted into cartilagedefective mice. The transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed only noncartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat possess the strong proliferative characteristic of stem cells and retain their ability to differentiate into hyaline cartilage after transplantation. On the contrary, cells cultured in a 1-G environment do not maintain these features. Simulated microgravity may thus provide an environment to successfully expand stem cell populations in vitro without culture supplements that can adversely affect stem cell-derived transplantations. This method has significant potential for regenerative medicine and developmental biology.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed7&AN=2007051139
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1089%2Fscd.2006.15.921&issn=1547-3287&isbn=&volume=15&issue=6&spage=921&pages=921-929&date=2006&title=Stem+Cells+and+Development&atitle=Microgravity+potentiates+stem+cell+proliferation+while+sustaining+the+capability+of+differentiation&aulast=Yuge&pid=%3Cauthor%3EYuge+L.%3C%2Fauthor%3E&%3CAN%3E2007051139%3C%2FAN%3E

Simulated Microgravity Maintains the Undifferentiated State and Enhances the Neural Repair Potential of Bone Marrow Stromal Cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Recently, regenerative medicine with bone marrow stromal cells (BMSCs) has gained significant attention for the treatment of central nervous system diseases. Here, we investigated the activity of BMSCs under simulated microgravity conditions. Mouse BMSCs (mBMSCs) were isolated from C57BL/6 mice and harvested in 1G condition. Subjects were divided into 4 groups: cultured under simulated microgravity and 1G condition in growth medium and neural differentiation medium. After 7 days of culture, the mBMSCs were used for morphological analysis, reverse transcription (RT)-polymerase chain reaction, immunostaining analysis, and grafting. Neural-induced mBMSCs cultured under 1G conditions exhibited neural differentiation, whereas those cultured under simulated microgravity did not. Moreover, under simulated microgravity conditions, mBMSCs could be cultured in an undifferentiated state. Next, we intravenously injected cells into a mouse model of cerebral contusion. Graft mBMSCs cultured under simulated microgravity exhibited greater survival in the damaged region, and the motor function of the grafted mice improved significantly. mBMSCs cultured under simulated microgravity expressed CXCR4 on their cell membrane. Our study indicates that culturing cells under simulated microgravity enhances their survival rate by maintaining an undifferentiated state of cells, making this a potentially attractive method for culturing donor cells to be used in grafting.

Related URLs:
<Go to ISI>://WOS:000290255300013

Detrimental effects of microgravity on mouse preimplantation development in vitro

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (microG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under microG. However, although we obtained 75 healthy offspring from microG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under microG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under microG environment in a mammal, but normal preimplantation embryo development might require 1G.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/19707597

Effects of simulated microgravity on embryonic stem cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed10&AN=2011698685
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1371%2Fjournal.pone.0029214&issn=1932-6203&isbn=&volume=6&issue=12&spage=e29214&pages=&date=2011&title=PLoS+ONE&atitle=Effects+of+simulated+microgravity+on+embryonic+stem+cells&aulast=Wang&pid=%3Cauthor%3EWang+Y.%3C%2Fauthor%3E&%3CAN%3E2011698685%3C%2FAN%3E

A Major Effect of Simulated Microgravity on Several Stages of Preimplantation Mouse Development is Lethality Associated With Elevated Phosphorylated SAPK/JNK

by cfynanon 9 June 2015in Biology & Biotechnology No comment

We tested whether microgravity affects mouse development during a period when gravity cues chick and frog embryo development. A rotating vessel developed similar to 0.1% simulated microgravity (MGS) for embryos. Microgravity simulation resulted in blocked cell accumulation in E2.5 embryos. E1.5 and E3.5 embryos showed lesser effects. For E1.5/2.5 embryos, cell accumulation block was followed by lethality at 48 hours after MGS. For E3.5 embryos, MGS blocked development without lethality but with apoptosis. E1.5-3.5 embryos from the rotational control developed lesser effects than MGS embryos. Embryonic stress-activated protein kinase (SAPK) was phosphorylated during MGS and mediated apoptosis. Increased pSAPK suggested that lethality is due to cellular stress induced by MGS, unlike the dysfunctional development after gravitational disorientation in frog and chick embryos. Thus, MGS causes lethality, a novel phenotype not often observed in microgravity or MGS. Embryonic lethality at E2.5 and apoptosis at E3.5 are associated with SAPK function, suggesting that MGS causes a general stress response that immediately affects many aspects of development. In addition, MGS and many aspects of In vitro fertilization/assisted reproductive technologies (IVF/ART) produce nonphysiological, nonevolutionary stresses that are mediated by SAPK, suggesting the primacy of this protein kinase in a wide range of mechanisms mediating negative reproductive outcomes in IVF/ART and potentially in spaceflight.

Related URLs:
<Go to ISI>://WOS:000270272600003

Rotating Microgravity-Bioreactor Cultivation Enhances the Hepatic Differentiation of Mouse Embryonic Stem Cells on Biodegradable Polymer Scaffolds

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Embryonic stem (ES) cells are pluripotent cells that are capable of differentiating all the somatic cell lineages, including those in the liver tissue. We describe the generation of functional hepatic-like cells from mouse ES (mES) cells using a biodegradable polymer scaffold and a rotating bioreactor that allows simulated microgravity. Cells derived from ES cells cultured in the three-dimensional (3D) culture system with exogenous growth factors and hormones can differentiate into hepatic-like cells with morphologic characteristics of typical mature hepatocytes. Reverse-transcription polymerase chain-reaction testing, Western blot testing, immunostaining, and flow cytometric analysis show that these cells express hepatic-specific genes and proteins during differentiation. Differentiated cells on scaffolds further exhibit morphologic traits and biomarkers characteristic of liver cells, including albumin production, cytochrome P450 activity, and low-density lipoprotein uptake. When these stem cell-bearing scaffolds are transplanted into severe combined immunodeficient mice, the 3D constructs remained viable, undergoing further differentiation and maturation of hepatic-like cells in vivo. In conclusion, the growth and differentiation of ES cells in a biodegradable polymer scaffold and a rotating microgravity bioreactor can yield functional and organizational hepatocytes useful for research involving bioartificial liver and engineered liver tissue.

Related URLs:
<Go to ISI>://WOS:000310752900019

Single cell and tissue specific methods for evaluation of radiation and microgravity effects

by cfynanon 9 June 2015in Biology & Biotechnology No comment

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=med4&AN=10631338
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:medline&id=pmid:10631338&id=doi:&issn=0027-5107&isbn=&volume=430&issue=2&spage=235&pages=235-40&date=1999&title=Mutation+Research&atitle=Single+cell+and+tissue+specific+methods+for+evaluation+of+radiation+and+microgravity+effects.&aulast=Van+Oostveldt&pid=%3Cauthor%3EVan+Oostveldt+P%3C%2Fauthor%3E&%3CAN%3E10631338%3C%2FAN%3E

Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption

by cfynanon 9 June 2015in Biology & Biotechnology No comment

During space flight, severe losses of bone mass are observed. Both bone formation and resorption are probably involved, but their relative importance remains unclear. The purpose of this research is to understand the role of osteoclasts and their precursors in microgravity-induced bone loss. Three experiments on isolated osteoclasts (OCs) and on their precursors, OSTEO, OCLAST, and PITS, were launched in the FOTON-M3 mission. The OSTEO experiment was conducted for 10 d in microgravity within bioreactors with a perfusion system, where the differentiation of precursors, cultured on a synthetic 3-dimensional bonelike biomaterial, skelite, toward mature OCs was assessed. In OCLAST and in PITS experiments, differentiated OCs were cultured on devitalized bovine bone slices for 4 d in microgravity. All of the experiments were replicated on ground in the same bioreactors, and OCLAST also had an inflight centrifuge as a control. Gene expression in microgravity, compared with ground controls, demonstrated a severalfold increase in genes involved in osteoclast maturation and activity. Increased bone resorption, proved by an increased amount of collagen telopeptides released VS ground and centrifuge control, was also found. These results indicate for the first time osteoclasts and their precursors as direct targets for microgravity and mechanical forces.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=medl&AN=19329761
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:medline&id=pmid:19329761&id=doi:10.1096%2Ffj.08-127951&issn=0892-6638&isbn=&volume=23&issue=8&spage=2549&pages=2549-54&date=2009&title=FASEB+Journal&atitle=Microgravity+during+spaceflight+directly+affects+in+vitro+osteoclastogenesis+and+bone+resorption.&aulast=Tamma&pid=%3Cauthor%3ETamma+R%3C%2Fauthor%3E&%3CAN%3E19329761%3C%2FAN%3E

Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in the International Space Station (ISS)

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/22438896
http://www.plosone.org/article/fetchObject.action?uri=info:doi/10.1371/journal.pone.0033179&representation=PDF

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