Research Containing: Bone marrow

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.

DECREASE IN THE NUMBER OF PROGENITORS OF ERYTHROCYTES (BFUE, CFU-E), GRANULOCYTES AND MACROPHAGES (GM-CFC) IN BONE-MARROW OF RATS AFTER A 14-DAY FLIGHT ONBOARD THE COSMOS-2044 BIOSATELLITE

by cfynanon 9 June 2015in Biology & Biotechnology No comment

A decrease in the number of progenitors of erythrocytes (BFUe, CFUe) and of granulocytes and macrophages (GM-CFC) in bone marrow was found in rats exposed to microgravitation during a 14-day flight onboard the Cosmos-2044 biosatellite when compared to control rats maintained under conditions of gravitation on the ground. The number of progenitors of both lineages of haemopoiesis was also decreased in synchronous control rats, thus suggesting that the pool of progenitors is influenced also by the action of the nonspecific space flight factors.

Related URLs:
<Go to ISI>://WOS:A1991FN07900005

Bone marrow fat accumulation after 60 days of bed rest persisted 1 year after activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Immobility in bed and decreased mobility cause adaptations to most human body systems. The effect of immobility on fat accumulation in hemopoietic bone marrow has never been measured prospectively. The reversibility of marrow fat accumulation and the effects on hemopoiesis are not known. In the present study, 24 healthy women (age: 25–40 yr) underwent −6° head-down bed rest for 60 days. We used MRI to noninvasively measure the lumbar vertebral fat fraction at various time points. We also measured hemoglobin, erythropoietin, reticulocytes, leukocytes, platelet count, peripheral fat mass, leptin, cortisol, and C-reactive protein during bed rest and for 1 yr after bed rest ended. Compared with baseline, the mean (± SE) fat fraction was increased after 60 days of bed rest (+2.5 ± 1.1%, P < 0.05); the increase persisted 1 yr after the resumption of regular activities (+2.3 ± 0.8%, P < 0.05). Mean hemoglobin levels were significantly decreased 6 days after bed rest ended (−1.36 ± 0.20 g/dl, P < 0.05) but had recovered at 1 yr, with significantly lower mean circulating erythropoietin levels (−3.8 ± 1.2 mU/ml, P < 0.05). Mean numbers of neutrophils and lymphocytes remained significantly elevated at 1 yr (+617 ± 218 neutrophils/μl and +498 ± 112 lymphocytes/μl, both P < 0.05). These results constitute direct evidence that bed rest irreversibly accelerated fat accumulation in hemopoietic bone marrow. The 2.5% increase in fat fraction after 60 days of bed rest was 25-fold larger than expected from historical ambulatory controls. Sixty days of bed rest accelerated by 4 yr the normal bone marrow involution. Bed rest and marrow adiposity were associated with hemopoietic stimulation. One year after subjects returned to normal activities, hemoglobin levels were maintained, with 43% lower circulating erythropoietin levels, and leukocytes remained significantly elevated across lineages. Lack of mobility alters hemopoiesis, possibly through marrow fat accumulation, with potentially wide-ranging clinical consequences.

Ex Vivo Expansion of Human Umbilical Cord Blood Hematopoietic Stem/Progenitor Cells with Support of Microencapsulated Rabbit Mesenchymal Stem Cells in a Rotating Bioreactor

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Expansion of human umbilical cord blood mononuclear cells (MNCs) was carried out with/without the support of alginate-chitosan-alginate (ACA) microcapsules containing rabbit bone marrow (rBM) mesenchymal stem cells (MSCs). Cells were cultured in a rotating wall vessel (RWV) bioreactor and also in tissue-culture plates using serum-free media supplemented with conventional doses of purified human recombinant cytokines for 7 days. The total nucleated cell density, pH and osmolality of the culture media in both co-culture systems were measured every 24 hours. Flow cytometry analysis of the CD34(+) population and methylcellulose colony assays for assessing the pluripotency of the population were carried out after Oh, 72h and 168h of culture. The RWV bioreactor co-culture, combined with a cell-dilution feeding protocol, was observed to be efficient in expanding UCB MNCs. By the end of 168h of culture using this system, the total nucleated cell number had grown around 107-fold, whilst the CD34(+) cells 26-fold and colony-forming units in culture 19-fold. Within RWV alone control and static co-culture control groups, however, expansions of total nucleated cell number were 52-fold and 10-fold, respectively, while CD34(+) cells and CFU-Cs numbers both changed mildly (p < 0.01, compared with RWV co-culture group). It was thus demonstrated that the expansion of HSCs can be achieved at a large-scale with the support of microencapsulated stromal cells using this bioreactor.

Related URLs:
<Go to ISI>://WOS:000291289600009

Selected Contribution: Effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions

by cfynanon 9 June 2015in Biology & Biotechnology No comment

There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34+ population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.

Detection of the quantity of kinesin and microgravity-sensitive kinesin genes in rat bone marrow stromal cells grown in a simulated microgravity environment

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Kinesin and kinesin-like proteins (KLPs) constitute a superfamily of microtubule motor proteins found in all eukaryotic organisms. Members of the kinesin superfamily are known to play important roles in many fundamental cellular and developmental processes. To date, few published studies have reported on the effects of microgravity on kinesin expression. In this paper, we describe the expression pattern and microgravity-sensitive genes of kinesin in rat bone marrow stromal cells cultured in a ground-based rotating bioreactor. The quantity of kinesin under the clinorotation condition was examined by immunoblot analysis with anti-kinesin. Furthermore, the distribution of kinesin at various times during clinorotation was determined by dual immunostaining, using anti-kinesin monoclonal antibody or anti-beta-tubulin monoclonal antibody. In terms of kinesin quantity, we found that the ratios of the amounts of clinorotated/stationary KLPs decreased from clinorotation day 5 to day 10, although it increased on days 2 and 3. Immunofluorescence analysis revealed that kinesin in the nucleus was the first to be affected by simulated microgravity, following the kinesin at the periphery that was affected at various times during clinorotation. Real-time RT-PCR analysis of kinesin mRNA expression was performed and led to the identification of 3 microgravity-sensitive kinesin genes: KIF9, KIFC1, and KIF21A. Our results suggest that kinesin has a distinct expression pattern, and the identification of microgravity-sensitive kinesin genes offers insight into fundamental cell biology. (C) 2010 Elsevier Ltd. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000291173900010

Activation of Nervous System Development Genes in Bone Marrow Derived Mesenchymal Stem Cells Following Spaceflight Exposure

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity-induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA-8+Increment 13) from March 30 to April 8, 2006 (experiment "Stroma-2''). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo-inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell-cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC. J. Cell. Biochem. 111: 442-452, 2010. (C) 2010 Wiley-Liss, Inc.

Related URLs:
<Go to ISI>://WOS:000282482400020

Ex vivo expansion of human umbilical cord blood hematopoietic stem/progenitor with support of microencapsulated rabbit mesenchymal stem cells in rotating wall vessel

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Expansion of human umbilical cord blood mononuclear cells (MNCs) was carried out with/without the support of alginate-chitosan-alginate (ACA) microcapsules containing rabbit bone marrow (rBM) mesenchymal stem cells (MSCs). Cells were cultured in a rotating wall vessel (RWV) bioreactor and also in tissue-culture plates using serum-free media supplemented with conventional doses of purified human recombinant cytokines for 7 days. The total nucleated cell density, pH and osmolality of the culture media in both co-culture systems were measured every 24 hours. Flow cytometry analysis of the CD34+ population and methylcellulose colony assays for assessing the pluripotency of the population were carried out after 0h, 72h and 168h of culture. The RWV bioreactor co-culture, combined with a cell-dilution feeding protocol, was observed to be efficient in expanding UCB MNCs. By the end of 168h of culture using this system, the total nucleated cell number had grown around 107-fold, whilst the CD34+ cells 26-fold and colony-forming units in culture 19-fold. Within RWV alone control and static co-culture control groups, however, expansions of total nucleated cell number were 52-fold and 10-fold, respectively, while CD34+ cells and CFU-Cs numbers both changed mildly (p < 0.01, compared with RWV co-culture group). It was thus demonstrated that the expansion of HSCs can be achieved at a large-scale with the support of microencapsulated stromal cells using this bioreactor.

Related URLs:
<Go to ISI>://WOS:000248035500193

Regulatory cross talks of bone cells, hematopoietic stem cells and the nervous system maintain hematopoiesis

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Adult hematopoietic stem cells (HSC) continuously replenish the blood with immune and blood cells with a finite life span, from the bone marrow (BM) reservoir of immature and maturing leukocytes. Regulation of HSC migration and development is essential for their function and blood cell production. These diverse and multiple states require a tight regulation to efficiently address host defense and repair requirements. Numerous recent studies disclose a central role for bone related cells in regulation of HSC and hematopoiesis. During ontogeny HSC home and seed the fetal BM in the last gestation period when the bone is already ossified. Ossification involves bone forming osteoblast- and bone degrading osteoclast activity and is considered essential for the formation of BM cavities and hematopoiesis. This synchronized association implies the need for active bone cells and bone turnover for HSC regulation. Osteoblastic cells and SDF- 1+/nestin+ reticular adventitial and CAR cells are crucial for regulation of HSC lodgment, self-renewal and function. Bone resorbing osteoclasts regulate bone turnover and progenitor cell detachment and release from the BM. Sympathetic signals from the nervous system activated by circadian rhythms or stress conditions control both bone turnover and HSC migration and development. In this review we discuss pathways and mechanisms involved in this orchestrated regulatory network. A special focus is made on the pivotal role of the SDF-1/CXCR4 axis as a determinant of HSC fate. Inflammation, DNA damage, cytokine mobilization, microgravity and aging are discussed as specific physiologic and pathologic events entailing dysregulation of the tightly balanced Bone-Brain-Blood triad.

Three-dimensional bioreactor cultures: A useful dynamic model for the study of cellular interactions

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The ex vivo expansion of hematopoietic cells is a developing area with emphasis on bioreactor systems for amelioration of culture conditions. A rational design of bioreactors, especially those allowing microgravity, could permit the production of stem cells and will offer new approaches for studying the mechanisms of proliferation, differentiation, and signal transduction of cultured cells. The efficacy of two commercially available bioreactors (rotating-vessel miniPERM and static INTEGRA CL 350) to support long-term bone marrow cell cultures (LTBMCC) and three-dimensional growth of Hodgkin's lymphoma HD-MY-Z cells was investigated. In the miniPERM system, the growth of LTBMCC spheroids (containing 30-40 cells) was obtained. An essentially higher content of hematopoietic precursor cells (colony-forming units-granulocyte macrophage) was registered in the rotating-vessel system. In this bioreactor, a growth of large HD-MY-Z spheroids (containing 100-200 cells) was achieved. The composed mathematical models of the physicomechanical behavior of spheroids enabled the evaluation of the revolution frequency increase schedule. The differential equations took into account all inertial effects caused by the production module rotation movement as well as those caused by the relative movement of the spheroid in the fluid. The models aimed at the optimization of the rotation frequency increase schedule for different types of cells to reduce shear stress, augment productivity, and tolerate the growth of large spheroids. The models were numerically tested using MATLAB-SIMULINK software, and the trajectories of prestained HD-MY-Z spheroids were filmed. The coincidence of the theoretical and experimental trajectories was sufficient.

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Research Containing: Bone marrow

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

DECREASE IN THE NUMBER OF PROGENITORS OF ERYTHROCYTES (BFUE, CFU-E), GRANULOCYTES AND MACROPHAGES (GM-CFC) IN BONE-MARROW OF RATS AFTER A 14-DAY FLIGHT ONBOARD THE COSMOS-2044 BIOSATELLITE

Bone marrow fat accumulation after 60 days of bed rest persisted 1 year after activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study

Ex Vivo Expansion of Human Umbilical Cord Blood Hematopoietic Stem/Progenitor Cells with Support of Microencapsulated Rabbit Mesenchymal Stem Cells in a Rotating Bioreactor

Selected Contribution: Effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions

Detection of the quantity of kinesin and microgravity-sensitive kinesin genes in rat bone marrow stromal cells grown in a simulated microgravity environment

Activation of Nervous System Development Genes in Bone Marrow Derived Mesenchymal Stem Cells Following Spaceflight Exposure

Ex vivo expansion of human umbilical cord blood hematopoietic stem/progenitor with support of microencapsulated rabbit mesenchymal stem cells in rotating wall vessel

Regulatory cross talks of bone cells, hematopoietic stem cells and the nervous system maintain hematopoiesis

Three-dimensional bioreactor cultures: A useful dynamic model for the study of cellular interactions