Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45^-CD34⁺CD105⁺ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31⁺ cells, CD31^- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31⁺ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31^- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal components were up-regulated. CD31⁺ cells did not proliferate in vitro. CD45^-CD34⁺CD105⁺CD31^- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.

The increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBA predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through ¹³C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.

Key Points: Eight extracellular biomarkers define the metabolic age of stored RBCs. Metabolomics defines a universal signature of RBC storage lesion.

A series of water-soluble cationic chitosan derivatives were prepared by chemoselective functionalization at the amino group of five different parent chitosans having varying degrees of acetylation and molecular weight. The quaternary moieties were introduced at different alkyl spacer lengths from the polymer backbone (C-0, C-2 and C-6) with the aid of 3,6-di-O-tert-butyldimethylsilyl protection of the chitosan backbone, thus allowing full (100%) substitution of the free amino groups. All of the derivatives were characterized using ¹H-NMR, ¹H-¹H COSY and FT-IR spectroscopy, while molecular weight was determined by GPC. Antibacterial activity was investigated against Gram positive S. aureus and Gram negative E. coli. The relationship between structure and activity/toxicity was defined, considering the effect of the cationic group’s structure and its distance from the polymer backbone, as well as the degree of acetylation within a molecular weight range of 7–23 kDa for the final compounds. The N,N,N-trimethyl chitosan with 100% quaternization showed the highest antibacterial activity with moderate cytotoxicity, while increasing the spacer length reduced the activity. Trimethylammoniumyl quaternary ammonium moieties contributed more to activity than 1-pyridiniumyl moieties. In general, no trend in the antibacterial activity of the compounds with increasing molecular weight or degree of acetylation up to 34% was observed.

Grafting of anoplin to chitosan polymers in varying density and orientation leads to a 30-fold activity enhancement and a reduction in toxicity towards red blood cell of more than two orders of magnitude.