Recovery of Human Mesenchymal Stem Cells Following Dehydration and Rehydration

Gordon, Stephen L.; Oppenheimer, Stephanie R.; Mackay, Alastair M.; Brunnabend, Jason; Puhlev, Iskren; Levine, Fred

doi:10.1006/cryo.2001.2361

Cited by 56 publications

(25 citation statements)

References 12 publications

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“…human primary fibroblasts provisioned with intracellular trehalose (52), but a subsequent report from the same group indicated that those water content analyses apparently were inaccurate (53). A group 3 LEA protein from the nematode Aphelenchus avenae transfected into the human cell line T-REx293 (derived from the embryonic kidney cell line HEK293) improved the tolerance to hyperosmotic shock induced by exposure to 100 mM NaCl or 400 mM sugars, but exposure of cells to air drying over periods of hours at different relative humidity levels resulted in mortality (54).…”

Section: Discussionmentioning

confidence: 99%

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Chakraborty

Borcar

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H 2 O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.water stress | biopreservation | intrinsically disordered proteins | osmolyte | Artemia franciscana

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Section: Discussionmentioning

confidence: 99%

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Chakraborty

Borcar

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Evidence indicates that the presence of small stress proteins and Late Embryogenesis Abundant (LEA) 2 proteins is also important for cellular protection during drying in eukaryotic cells (13)(14)(15)(16). However, the issue of how subcellular compartments in nucleated cells are protected during water stress has received far less attention (17)(18)(19)(20), and this lack of understanding may explain our limited success in stabilizing eukaryotic cells from nontolerant species (21)(22)(23)(24)(25)(26)(27). We report here the novel targeting of a LEA protein to the mitochondrial compartment of an animal species and the associated protection against water stress.…”

mentioning

confidence: 99%

Occurrence of Mitochondria-targeted Late Embryogenesis Abundant (LEA) Gene in Animals Increases Organelle Resistance to Water Stress

Menze

Boswell

Toner

et al. 2009

Journal of Biological Chemistry

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Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shrimp Artemia franciscana is 34 kDa. Bioinformatic analyses reveal features typical of a Group 3 LEA protein, and subcellular localization programs predict targeting of the mature peptide to the mitochondrial matrix, based on an N-terminal, amphipathic presequence. Real-time quantitative PCR shows that Afralea3m mRNA is expressed manyfold higher in desiccation-tolerant embryonic stages when compared with intolerant nauplius larvae. Mitochondrial localization of the protein was confirmed by transfection of human hepatoma cells (HepG2/C3A) with a nucleotide construct encoding the first 70 N-terminal amino acids of AfrLEA3m in-frame with the nucleotide sequence for green fluorescence protein. The chimeric protein was readily incorporated into mitochondria of these cells. Successful targeting of a protein to human mitochondria by use of an arthropod signaling sequence clearly reveals the highly conserved nature of such presequences, as well as of the import machinery. Finally, mitochondria isolated from A. franciscana embryos, which naturally contain AfrLEA3m and trehalose, exhibit resistance to water stress (freezing) as evidenced by an unchanged capacity for oxidative phosphorylation on succinate ؉ rotenone, a resistance that is absent in mammalian mitochondria lacking AfrLEA3m.Fluctuation in cellular water content is a universal problem confronting a variety of organisms, and any environmental stress that impacts cellular water poses a risk to life (1). Nevertheless, some animals are able to cope with virtually complete loss of cellular water for prolonged times (2, 3), a phenomenon termed anhydrobiosis. Tolerance to water stress (i.e. evaporative water loss, freezing, and osmotic removal of water) is most likely achieved by several different mechanisms designed to protect and repair the structures and functions of cells and tissues. Many desiccation-tolerant organisms respond to water stress by intracellular accumulation of selected sugars, amino acids and derivatives, and methylamines (often occurring with urea) termed "compatible" osmolytes (4 -7). Organic solutes such as the non-reducing sugar trehalose can actually stabilize biological structures during severe drying (8 -12). Evidence indicates that the presence of small stress proteins and Late Embryogenesis Abundant (LEA) 2 proteins is also important for cellular protection during drying in eukaryotic cells (13)(14)(15)(16). However, the issue of how subcellular compartments in nucleated cells are protected during water stress has received far less attention (17)(18)(...

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“…Small living cells have been genetically engineered to synthesize trehalose endogenously. This approach requires the constant production of adenoviral vectors that exhibit significant cytotoxicity, particularly at high multiplicities of infection (Gordon et al 2001;Guo et al 2000;Puhlev et al 2001). Trehalose has also been introduced into mammalian cells or their organelles through engineered or native transmembrane pores Chen et al 2001b;Elliott et al 2006;Eroglu et al 2000;Liu et al 2005), electroporation (Reuss et al 2004;Shirakashi et al 2002), fluid-phase endocytosis (He et al 2006a;Oliver 2004;Wolkers et al 2003), and lipid phase transition (Beattie et al 1997;He et al 2006a).…”

Section: Freeze-dryingmentioning

confidence: 99%

Preservation of Embryonic Stem Cells

He¹

2011

Methodological Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells for Basic and Practical Applicatio

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Recovery of Human Mesenchymal Stem Cells Following Dehydration and Rehydration

Cited by 56 publications

References 12 publications

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Occurrence of Mitochondria-targeted Late Embryogenesis Abundant (LEA) Gene in Animals Increases Organelle Resistance to Water Stress

Preservation of Embryonic Stem Cells

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