Using stem and progenitor cells to recapitulate kidney development and restore renal function.

PURPOSE OF REVIEW: There is considerable interest in the idea of generating stem and precursor cells that can differentiate into kidney cells and be used to treat kidney diseases. Within this field, we highlight recent research articles focussing on mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and kidney-derived stem/progenitor cells (KSPCs). RECENT FINDINGS: In preclinical studies, MSCs ameliorate varied acute and chronic kidney diseases. Their efficacy depends on immunomodulatory and paracrine properties but MSCs do not differentiate into functional kidney epithelia. iPSCs can be derived from healthy individuals and from kidney patients by forced expression of precursor genes. Like ESCs, iPSCs are pluripotent and so theoretically they have the potential to form functional kidney epithelia when used therapeutically. KSPCs, existing as cell subsets within adult and developing kidneys, constitute attractive future therapeutic agents. SUMMARY: Results from preclinical studies are encouraging but caution is required regarding potential human therapeutic applications because molecular, morphological and functional characterization of 'kidney cells' generated from ECSs, iPSCs, KSPCs have not been exhaustive. The long-term safety of renal stem and precursor cells needs more study, including potential negative effects on renal growth and their potential for tumor formation.

Leukemia inhibitory factor promotes Hsp90 association with STAT3 in mouse embryonic stem cells.

Self-renewal of in vitro cultured mouse embryonic stem (mES) cells is dependent on the presence of leukemia inhibitory factor (LIF). LIF induces overexpression and tyrosine phosphorylation of STAT3 (signal transducer and activator of transcription 3) and its subsequent nuclear translocation. The molecular chaperone heat shock protein 90 (Hsp90) is involved in the activation and maturation of a wide variety of substrate proteins. We investigated the effect of LIF withdrawal on the protein expression levels of STAT3 and Hsp90 and on the interactions between STAT3 and Hsp90. Taken together the data presented here suggest that LIF promotes the interaction of Hsp90 with STAT3 during self-renewal, indicating a potentially pivotal role for Hsp90 in the LIF-based maintenance of self-renewal of mouse embryonic stem cells.

Beyond the lipid hypothesis: mechanisms underlying phenotypic plasticity in inducible cold tolerance.

The physiological adjustment of organisms in response to temperature variation is a crucial part of coping with environmental stress. An important component of the cold response is the increase in membrane lipid unsaturation, and this has been linked to an enhanced resistance to the debilitating or lethal effects of cold. Underpinning the lipid response is the upregulation of fatty acid desaturases (des), particularly those introducing double bonds at the 9-10 position of saturated fatty acids. For plants and microbes there is good genetic evidence that regulation of des genes, and the consequent changes in lipid saturation, are causally linked to generation of a cold-tolerant phenotype. In animals, however, supporting evidence is almost entirely limited to correlations of saturation with cold conditions. We describe our recent attempts to provide a direct test of this relationship by genetic manipulation of the nematode Caenorhabditis elegans. We show that this species displays a strong cold tolerant phenotype induced by prior conditioning to cold, and that this is directly linked to upregulated des activity. However, whilst genetic disruption of des activity and lipid unsaturation significantly reduced cold tolerance, animals retained a substantial component of their stress tolerant phenotype produced by cold conditioning. This indicates that mechanisms other than lipid unsaturation play an important role in cold adaptation.

Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells.

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and Results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

Characterisation of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori.

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29) and C(28)) yielding cholesterol (C(27)). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

Serum and Gingival Fluid Antibodies as Adjuncts in the Diagnosis of Actinobacillus actinomycetemcomitans Associated Periodontal Disease.

Serum antibody titers to Actinobacillus actinomycetemcomitans were measured in 200 subjects by an enzyme-lined immunosorbent assay (ELISA) using whole microorganisms as antigen. Comparisons were made between titers found in periodontally normal subjects and titers in subjects with localized juvenile periodontitis (LJP), postlocalized juvenile periodontitis, generalized juvenile periodontitis or adult periodontitis. It was found that titers to all three serotypes of A. actinomycetemcomitans were elevated in LJP patients' sera, while serum antibody levels in other diseased groups were not significantly elevated to any of the serotypes. Patient sera were also examined for serum antibody to oral Haemophili previously shown to cross-react with A. actinomycetemcomitans. Similar antibody titers were found in both normal subjects and in patients with various forms of periodontal disease to Haemophilus aphrophilus, H. influenzae and H. parainfluenzae. The A. actinomycetemcomitans antibodies which were elevated in LJP patients could not be correlated with antibody titers to cross-reacting Haemophili, suggesting that these antibodies are A. actinomycetemcomitansspecific. Serum antibody responses in six of the LJP patients were assessed to autologous strains of A. actinomycetemcomitans. Each patient was found to be infected with only a single serotype of A. actinomycetemcomitans, and specific antibodies to the infecting serotype were found in the patients' sera. In families, the LJP patients had significantly elevated IgG, IgA and IgM serum antibody titers to A actinomycetemcomitans, while the IgG and IgA antibody titers in periodontally normal siblings were at levels comparable to those found in normal subjects. However, IgM serum antibodies were elevated in the periodontally normal siblings of LJP patients suggesting that the formation of IgM antibodies to A. actinomycetemcomitans may precede the clinical appearance of localized juvenile periodontitis. Gingival crevicular fluid and serum antibody levels to A. actinomycetemcomitans were compared in LJP patients. Comparable titers of IgG, IgA and IgM antibodies were found in serum and gingival fluid in most subjects; however, gingival fluid samples sometimes showed higher titers than serum, likely resulting from local antibody synthesis. The value of serum antibody determinations to A. actinomycetemcomitans in the diagnosis of Actinobacillus-associated periodontitis was also assessed. The predictive value of a positive test (significantly elevated anti-A actinomycetemcomitans IgG) was 86%, while the specificity was 89%. These results suggest that the measurement of serum or gingival crevicular fluid antibodies to A. actinomycetemcomitans may be valuable in the diagnosis of Actinobacillusassociated periodontal disease.