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Research Containing: Cell Proliferation

Plant cell proliferation and growth are altered by microgravity conditions in spaceflight

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/19864040
http://www.sciencedirect.com/science/article/pii/S0176161709003812

Production of stem cells with embryonic characteristics from human umbilical cord blood

by cfynanon 9 June 2015in Biology & Biotechnology No comment

When will embryonic stem cells reach the clinic? The answer is simple not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, – cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-I, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity-bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, (alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Related URLs:
<Go to ISI>://WOS:000231540000006

Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24 It. Flow cytometric assay for CD34(+) cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 It. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5 +/- 87.6 times, and CD34(+) cells 32.7 +/- 15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7 +/- 4.9 times. While in T-flasks, however, total cells density changed mildly, CD34(+) cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask. (c) 2006 Elsevier B.V. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000239156700012

[Expression of splice-variants of the insulin-like growth factor I and the condition of the cell-myosatellite pool in the rat m. soleus under conditions of gravitation relief and passive stretching]

by cfynanon 9 June 2015in Biology & Biotechnology No comment

We suggested that satellite proliferation with subsequent incorporation of their nuclei in the fibres was essential for enhancement of the protein synthesis in the rat relieved m. soleus. In passive stretch of the muscle, the number of labelled cells exceeded 2.5-fold their number in hung out animals and 1.7-fold in the control animals. The expression level of the insulin-like growth factor I (IF-1) was determined in the m. soleus after hanging out with stretching, and no changes were revealed as compared with hanging out without stretching in the control group. We believe that the muscle IF-1 and satellite cell incorporation are not essential for preventing atrophy of relieved soleus muscle in stretching.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=medl&AN=19899713
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:medline&id=pmid:19899713&id=doi:&issn=0869-8139&isbn=&volume=95&issue=9&spage=969&pages=969-76&date=2009&title=Rossiiskii+Fiziologicheskii+Zhurnal+Imeni+I.+M.+Sechenova&atitle=%5BExpression+of+splice-variants+of+the+insulin-like+growth+factor+I+and+the+condition+of+the+cell-myosatellite+pool+in+the+rat+m.+soleus+under+conditions+of+gravitation+relief+and+passive+stretching%5D.&aulast=Lomonosova&pid=%3Cauthor%3ELomonosova+IuN%3C%2Fauthor%3E&%3CAN%3E19899713%3C%2FAN%3E

Auxin transport and ribosome biogenesis mutant/reporter lines to study plant cell growth and proliferation under altered gravity

by cfynanon 9 June 2015in Biology & Biotechnology No comment

We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (µg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (µg RPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity.

Related URLs:

 Auxin transport and ribosome biogenesis mutant/reporter lines to study plant cell growth and proliferation under altered gravity

by cfynanon 9 June 2015in Biology & Biotechnology No comment

We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (µg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (µg RPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity.

Related URLs:

Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Faster growth and differentiation of liver stem cells to hepatocyte is one of the key factors during liver regeneration. In recent years, simulated microgravity, a physical force has shown to differentially regulate the differentiation and proliferation of stem cells. In the present work, we studied the effect of simulated microgravity on differentiation and proliferation of liver stem cells. The cells were subjected to microgravity, which was simulated using indigenously fabricated 3D clinostat. Proliferation, apoptosis, immunofluorescence assays and Western blot analysis were carried out to study the effects of simulated microgravity on liver stem cells. Microgravity treatment for 2 h enhanced proliferation of stem cells by twofold without inducing apoptosis and compromising cell viability. Analysis of hepatocyte nuclear factor 4-alpha (HNF4-alpha) expression after 2 h of microgravity treatment revealed that microgravity alone can induce the differentiation of stem cells within 2-3 days. Probing bone morphogenic protein 4 (BMP4) and Notch1 in microgravity treated stem cells elaborated downregulation of Notch1 and upregulation of BMP4 after 2 days of incubation. Further, blocking BMP4 using dorsomorphin and chordin conditioned media from chordin plasmid transfected cells attenuated microgravity mediated differentiation of liver stem cells. In conclusion, microgravity interplays with BMP4/Notch1 signaling in stem cells thus inducing differentiation of stem cells to hepatocytes. Present findings can be implicated in clinical studies where microgravity activated stem cells can regenerate the liver efficiently after liver injury.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed10&AN=2011438430
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1002%2Fjcb.23110&issn=0730-2312&isbn=&volume=112&issue=7&spage=1898&pages=1898-1908&date=2011&title=Journal+of+Cellular+Biochemistry&atitle=Simulated+microgravity+promoted+differentiation+of+bipotential+murine+oval+liver+stem+cells+by+modulating+BMP4%2FNotch1+signaling&aulast=Majumder&pid=%3Cauthor%3EMajumder+S.%3C%2Fauthor%3E&%3CAN%3E2011438430%3C%2FAN%3E

NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory'' environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Related URLs:
<Go to ISI>://WOS:000297350800010

NASA-approved rotary bioreactor enhances proliferation and osteogenesis of human periodontal ligament stem cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Previous studies have suggested that periodontal ligament stem cells (PDLSCs) play crucial role in regeneration of periodontal defects, and recently tissue engineering based on PDLSCs to enhance periodontal regeneration has been the focus of periodontal research. A theoretical way to achieve this goal would be to provide a "stimulatory'' environment to rapidly expand PDLSCs in vitro to expedite tissue engineering of periodontium. We hypothesize that three-dimensional (3D) dynamic simulated microgravity (SMG) culture system have effect on periodontal stem cells, and would benefit periodontal stem cells proliferation and differentiation, but up to now, there are no related reports on this aspect. In this study, we investigated the biological effect of three-dimensional dynamic SMG induced by rotary cell culture system (RCCS) on human periodontal ligament stem cells (hPDLSCs) in vitro. hPDLSCs were isolated from surgically extracted human teeth and enriched by collecting multiple colonies. hPDLSCs were inoculated on Cytodex 3 microcarriers and cultured in RCCS. The results showed that SMG affected the biology of hPDLSCs as indicated by promotion of proliferation and viability, alterations of morphology, and disorganization of microfilament system. Besides, SMG-treated hPDLSCs presented increased matrix mineralization and up-regulated expression of mineralization associated genes after incubation in osteogenic medium. For it is the first time to investigate effects of SMG on PDLSCs, the research may lend insight into variations of cell response in 3D environment, and contribute to achievement of desirable periodontal regeneration utilizing PDLSCs-based tissue engineering approaches.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=2009606938
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1089%2Fscd.2008.0371&issn=1547-3287&isbn=&volume=18&issue=9&spage=1273&pages=1273-1282&date=2009&title=Stem+Cells+and+Development&atitle=NASA-approved+rotary+bioreactor+enhances+proliferation+and+osteogenesis+of+human+periodontal+ligament+stem+cells&aulast=Li&pid=%3Cauthor%3ELi+S.%3C%2Fauthor%3E&%3CAN%3E2009606938%3C%2FAN%3E

Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-)4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=20386701
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:20386701&id=doi:&issn=1932-6203&isbn=&volume=5&issue=4&spage=e10035&pages=e10035&date=2010&title=PloS+one&atitle=Rapid+selection+and+proliferation+of+CD133%2B+cells+from+cancer+cell+lines%3A+chemotherapeutic+implications&aulast=Kelly&pid=%3Cauthor%3EKelly+S.E.%3C%2Fauthor%3E&%3CAN%3E20386701%3C%2FAN%3E

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