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Research Containing: RWV

Transient gene and microRNA expression profile changes of confluent human fibroblast cells in spaceflight

by on 22 August 2016in Biology & Biotechnology No comment

Microgravity, or an altered gravity environment different from the 1 g of the Earth, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies that have been conducted in space or by using simulated microgravity on the ground have focused on the growth or differentiation of these cells. It has not been specifically addressed whether nonproliferating cultured cells will sense the presence of microgravity in space. In an experiment conducted onboard the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 d, respectively, to investigate changes in gene and microRNA (miRNA) expression profiles in these cells. Results of the experiment showed that on d 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67(+) cells. Gene and miRNA expression data indicated activation of NF-kappaB and other growth-related pathways that involve hepatocyte growth factor and VEGF as well as the down-regulation of the Let-7 miRNA family. On d 14, when the cells were mostly nonproliferating, the gene and miRNA expression profile of the flight sample was indistinguishable from that of the ground sample. Comparison of gene and miRNA expressions in the d 3 samples, with respect to d 14, revealed that most of the changes observed on d 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for alpha-tubulin and fibronectin showed no difference between the flown and ground samples. Taken together, our study suggests that in true nondividing human fibroblast cells in culture, microgravity experienced in space has little effect on gene and miRNA expression profiles.-Zhang, Y., Lu, T., Wong, M., Wang, X., Stodieck, L., Karouia, F., Story, M., Wu, H. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in spaceflight.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/26917741

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 on 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

Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation

by on 9 June 2015in Biology & Biotechnology No comment

Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (mug) conditions. Scientists on the ground use two models of simulated mug (smug): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true mug when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true mug and smug on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2ralpha, Ifngamma, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that smug models provide an excellent test bed for scientists to develop baseline studies and augment true mug in spaceflight experiments. Ultimately, smug and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/25568077
http://ajpregu.physiology.org/content/308/6/R480

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

by on 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

Hematopoietic stem cells in microgravity

by on 9 June 2015in Biology & Biotechnology No comment

Engraftment and progenitor expansion was compared after microgravity or conventional culture. Rotating Wall Vessels (RWV) were compared with Teflon flask cultures. Two systems were used: 1) Male BALB/c whole bone marrow was cultured with varying cytokines, time intervals ranging from 24 hours to 7 days at 10–40 RPM. The cultured marrow was competed with an equal amount of fresh female marrow into lethally ablated female BALB/c mice. 2) Male B6.SJL (CD45.1) marrow was cultured with varying time intervals ranging from 24 hours to 14 days at 10 RPM with FLT-3 ligand, steel factor, and thrombopoietin. Fresh C57BL/6J (CD 45.2) marrow (3 × 106 cells) was competed with 3 × 106 cultured B6SJL male cells in lethally ablated male C57BL recipients. Engraftment was analyzed at 3–10 weeks. Progenitor expansion increased over controls by 24 hours with significantly greater expansion in the RWV cultures by 48 hours. The dominant theme was a fluctuation in engraftment phenotype, which transcended cytokine combination and murine strain. A nadir of engraftable stem cells occurred between 36–44 hours in Teflon flask cultures and 30–48 hours in RWV cultures. There was a consistent phase shift of engraftment between culturing conditions of 4–6 hours. The engraftable stem cells in RWV showed superior support of short-term engraftment in multiple experiments up to 32 hours in culture. Slower rational speed improved RWV engraftment and in-vitro progenitor number. Longer culturing time gave similar engraftment results. Surrogate progenitors from both culture conditions did not reflect engraftment data, with an overall increase in progenitors over time compared with dramatic engraftment fluctuation. Enhanced plating efficiency and increased number of progenitors seen in the RWV cultures when compared with the Teflon flask cultures persisting after 24 hours in culture. The fluctuating engraftment phenotype persists with different strains and cytokine combinations, signifying that this is a crucial physiologic phenomenon.

Related URLs:
<Go to ISI>://WOS:000088004200267