Research Containing: Cell Differentiation

Mechanical unloading of bone in microgravity reduces mesenchymal and hematopoietic stem cell-mediated tissue regeneration

by cfynanon 22 August 2016in Biology & Biotechnology No comment

Mechanical loading of mammalian tissues is a potent promoter of tissue growth and regeneration, whilst unloading in microgravity can cause reduced tissue regeneration, possibly through effects on stem cell tissue progenitors. To test the specific hypothesis that mechanical unloading alters differentiation of bone marrow mesenchymal and hematopoietic stem cell lineages, we studied cellular and molecular aspects of how bone marrow in the mouse proximal femur responds to unloading in microgravity. Trabecular and cortical endosteal bone surfaces in the femoral head underwent significant bone resorption in microgravity, enlarging the marrow cavity. Cells isolated from the femoral head marrow compartment showed significant down-regulation of gene expression markers for early mesenchymal and hematopoietic differentiation, including FUT1(-6.72), CSF2(-3.30), CD90(-3.33), PTPRC(-2.79), and GDF15(-2.45), but not stem cell markers, such as SOX2. At the cellular level, in situ histological analysis revealed decreased megakaryocyte numbers whilst erythrocytes were increased 2.33 fold. Furthermore, erythrocytes displayed elevated fucosylation and clustering adjacent to sinuses forming the marrow-blood barrier, possibly providing a mechanistic basis for explaining spaceflight anemia. Culture of isolated bone marrow cells immediately after microgravity exposure increased the marrow progenitor’s potential for mesenchymal differentiation into in-vitro mineralized bone nodules, and hematopoietic differentiation into osteoclasts, suggesting an accumulation of undifferentiated progenitors during exposure to microgravity. These results support the idea that mechanical unloading of mammalian tissues in microgravity is a strong inhibitor of tissue growth and regeneration mechanisms, acting at the level of early mesenchymal and hematopoietic stem cell differentiation.

Model microgravity enhances endothelium differentiation of mesenchymal stem cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Mesenchymal stem cells (MSCs) are capable of differentiation into multilineage cell types under certain induction conditions. Previous studies have demonstrated that physical environments and mechanical force can influence MSC fate, indicating that these factors may be favorable inducers for clinical treatment. Our previous study found that MSCs are spread with a spindle shape when cultured in normal gravity (NG), and under modeled microgravity (MMG) for 72 h, they become unspread and round and their cytoskeleton fibers are reorganized. These morphological changes affected the function of MSCs through the activity of RhoA. We examined the responses of MSCs under MMG stimulation, followed with VEGF differentiation. We found that MSCs under MMG for 72 h were differentiated into endothelial-like cells by detecting the expression of endothelial-specific molecules (Flk-1 and vWF), which were also able to form a capillary network. Their endothelial differentiation potential was improved under MMG compared with that under NG. We believe that this method is a novel choice of MMG stimulation for neovascularization. This phenomenon may increase the potential of MSC differentiation, which might be a new strategy for the treatment of various vascular diseases and improve vascularization in tissue engineering.

Related URLs:
<Go to ISI>://WOS:000314275500002

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation it) vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2x, p < 0.01), and maintained high cellular viability in the core, which was 2000 Inn from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5x P < 0.01), calcium deposition (5.5x, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2x, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications. (C) 2009 Elsevier Ltd. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000264953900005

Capacity of omega-3 fatty acids or eicosapentaenoic acid to counteract weightlessness-induced bone loss by inhibiting NF-kappaB activation: from cells to bed rest to astronauts

by cfynanon 9 June 2015in Biology & Biotechnology No comment

NF-kappaB is a transcriptional activator of many genes, including some that lead to muscle atrophy and bone resorption-significant concerns for astronauts. NF-kappaB activation is inhibited by eicosapentaenoic acid (EPA), but the influence of this omega-3 fatty acid on the effects of weightlessness are unknown. We report here cellular, ground analogue, and spaceflight findings. We investigated the effects of EPA on differentiation of RAW264.7 monocyte/macrophage cells induced by receptor activator of NF-kappaB ligand (RANKL) and on activation of NF-kappaB by tumor necrosis factor alpha (TNF-alpha) or exposure to modeled weightlessness. EPA (50 microM for 24 hours) inhibited RANKL-induced differentiation and decreased activation of NF-kappaB induced by 0.2 microg/mL of TNF-alpha for 30 minutes or by modeled weightlessness for 24 hours (p < .05). In human studies, we evaluated whether NF-kappaB activation was altered after short-duration spaceflight and determined the relationship between intake of omega-3 fatty acids and markers of bone resorption during bed rest and the relationship between fish intake and bone mineral density after long-duration spaceflight. NF-kappaB was elevated in crew members after short-duration spaceflight, and higher consumption of fish (a rich source of omega-3 fatty acids) was associated with reduced loss of bone mineral density after flight (p < .05). Also supporting the cell study findings, a higher intake of omega-3 fatty acids was associated with less N-telopeptide excretion during bed rest (Pearson r = -0.62, p < .05). Together these data provide mechanistic cellular and preliminary human evidence of the potential for EPA to counteract bone loss associated with spaceflight.

Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Mesenchymal stem cells (MSCs) are multi-potent, and the chondrogenesis of MSCs is affected by mechanical stimulation. The aim of this study was to investigate, using a rotary cell culture system (RCCS) bioreactor, the effects of microgravity on the chondrogenic differentiation of human adipose-derived MSCs (ADSCs), which were cultured in pellets with or without the chondrogenic growth factor TGF-beta1. In addition, we evaluated the role of the p38 MAPK pathway in this process. The real-time PCR and histological results show that microgravity has a synergistic effect on chondrogenesis with TGF-beta1. The p38 MAPK pathway was activated by TGF-beta1 alone and was further stimulated by microgravity. Inhibition of p38 activity with SB203580 suppressed chondrocyte-specific gene expression and matrix production. These findings suggest that the p38 MAPK signal acts as an essential mediator in the microgravity-induced chondrogenesis of ADSCs.

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

Simulated microgravity compromises mouse oocyte maturation by disrupting meiotic spindle organization and inducing cytoplasmic blebbing

by cfynanon 9 June 2015in Biology & Biotechnology No comment

In the present study, we discovered that mouse oocyte maturation was inhibited by simulated microgravity via disturbing spindle organization. We cultured mouse oocytes under microgravity condition simulated by NASA's rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation. While the rate of germinal vesicle breakdown did not differ between 1 g gravity and simulated microgravity, rate of oocyte maturation decreased significantly in simulated microgravity. The rate of maturation was 8.94% in simulated microgravity and was 73.0% in 1 g gravity. The results show that the maturation of mouse oocytes in vitro was inhibited by the simulated microgravity. The spindle morphology observation shows that the microtubules and chromosomes can not form a complete spindle during oocyte meiotic maturation under simulated microgravity. And the disorder of gamma-tubulin may partially result in disorganization of microtubules under simulated microgravity. These observations suggest that the meiotic spindle organization is gravity dependent. Although the spindle organization was disrupted by simulated microgravity, the function and organization of microfilaments were not pronouncedly affected by simulated microgravity. And we found that simulated microgravity induced oocytes cytoplasmic blebbing via an unknown mechanism. Transmission electron microscope detection showed that the components of the blebs were identified with the cytoplasm. Collectively, these results indicated that the simulated microgravity inhibits mouse oocyte maturation via disturbing spindle organization and inducing cytoplasmic blebbing.

Regulation of osteogenetic differentiation of mesenchymal stem cells by two axial rotational culture

by cfynanon 9 June 2015in Biology & Biotechnology No comment

It is crucial to understand how gravitational force affects the osteogenic differentiation of mesenchymal stem cells (MSCs), and these fundamental aspects hold promise for the development of a novel model of MSC regulation for cell proliferation and differentiation. The objective of this study was to investigate how significantly gravitational dispersion affects the spontaneously induced osteogenic differentiation of MSCs. Expression of surface antigen was measured by flow cytometry prior to two axial rotational cultures. About 12,500 hMSC cells were spread on culture wells of 1.8 cm(2) surface area and incubated for 7 days at 5% CO(2). The culture medium, 10% FCS/DMEM containing 3 ng/ml bFGF, was replaced every 3 days. Four wells then were placed in a 50-ml centrifugal tube filled with 10% FCS/DMEM without bFGF. The centrifugal tube was attached to the center of the rotor, and two axial rotational cultures were started at 10 rpm each of both rotational speeds. It was confirmed that the hMSCs used in this study expressed typical surface antigens as well as a multipotent differentiation ability for either osteogenic or adipogenic differentiation. Spontaneous expression of alkaline phosphatase (Alp) mRNA following the conventional static culture (1G condition) was suppressed by two axial rotational cultures for 7 days (p < 0.05). A separate study indicated that the cell count number eventually increased from 24,700 +/- A 6,400 to 78,400 +/- A 18,700 (p < 0.05). In addition, suppressed Alp mRNA was recovered after an additional 7-day culture under static conditions. This result indicated that dispersion of gravity is a promising modality to regulate osteogenic differentiation of hMSCs.

Related URLs:
<Go to ISI>://WOS:000297787000007

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.

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.

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Research Containing: Cell Differentiation

Mechanical unloading of bone in microgravity reduces mesenchymal and hematopoietic stem cell-mediated tissue regeneration

Model microgravity enhances endothelium differentiation of mesenchymal stem cells

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

Capacity of omega-3 fatty acids or eicosapentaenoic acid to counteract weightlessness-induced bone loss by inhibiting NF-kappaB activation: from cells to bed rest to astronauts

Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway

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

Simulated microgravity compromises mouse oocyte maturation by disrupting meiotic spindle organization and inducing cytoplasmic blebbing

Regulation of osteogenetic differentiation of mesenchymal stem cells by two axial rotational culture

Detrimental effects of microgravity on mouse preimplantation development in vitro

Effects of simulated microgravity on embryonic stem cells