Noninvasive sorting of stem cell aggregates based on intrinsic markers.

Noninvasive biomarkers hold important potential for the characterization and purification of stem cells because the addition of exogenous labels, probes, or reporters, as well as the disruption of cell-cell and cell-extracellular matrix interactions, can unintentionally but dramatically alter stem cell state. We recently showed that intensity of the intrinsically fluorescent metabolite, nicotinamide adenine dinucleotide (NADH), fluctuates predictably with changes in stem cell viability and differentiation state. Here, we use multiphoton flow cytometry developed in our laboratory to rapidly and noninvasively characterize and purify populations of intact stem cell aggregates based on NADH intensity and assessed the differentiation capacity of sorted populations. We found removal of aggregates with NADH intensity indicative of cell death resulted in a remaining population of aggregates significantly more likely to produce beating cardiomyocytes (26% vs. 8%, P < 0.05). Similarly, we found isolation of stem cell aggregates with NADH intensity indicative of future cardiac differentiation gave rise to more aggregates with beating cardiomyocytes at later time points (50% vs. 28%, P < 0.05). Further, coupling NADH intensity with gating based on size, enhances the enrichment for EBs capable of giving rise to cardiomyocytes (59% vs. 27%, P < 0.05). Thus, we demonstrate that endogenous properties of cell aggregates, such as NADH and size, can serve as gating parameters for large particle sorting devices to purify populations of stem cells or their progeny in a noninvasive manner, leading the way for improved therapeutic applications.

Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation.

Embryoid bodies (EBs) are large (>100 µm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 µm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.

Microfluidic sorting of microtissues.

Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function.

New approaches to replacing failing organs.

The foremost cause of death and disability is organ failure. Failure of the heart, lungs, kidney, and liver is typically addressed by organ transplantation; however, the number of human organs available for this purpose is quite limited. For this reason, some new technologies are being advanced for the treatment of organ failure. This communication summarizes potential limitations of these technologies.

Evaluation of the significance of dietary folate from wild vegetables in Vietnam.

Data on the overall dietary folate intakes among high-risk groups in poor countries is very limited. Vegetables are considered good sources but the evaluation of their contribution is hampered by the lack of data on folate concentrations in many traditional foods. Data on the analysis of folate concentrations in 16 wild vegetables used in the Mekong Delta and the Central Highlands in Vietnam and an evaluation of the relative importance of different foods in folate intakes of women is presented. Vegetable samples were collected in four study villages, blanched and frozen samples were transported to Sweden for analysis. Freeze-dried samples were analysed for total folate quantification using a commercial radio protein binding assay. Daily folate intakes among women were estimated from 7-day food frequency interviews with 213 women. The folate concentration in the vegetable samples ranged from 10 to 96 microg/100 g. The mean estimated daily folate intake among the 213 women in the study areas was 251 microg. Vegetables contributed approximately one-third of the daily folate intake, of which 72% and 42%, respectively, in the two regions was from wild vegetables. A majority of the women (87%) got some dietary folate from wild vegetables and nearly one-third had mean daily folate intakes of > 50 microg from such hidden food sources. The evaluation of dietary folate is complicated by data gaps in food composition tables, the unreliability of existing food data, variations between methods used for folate analysis and limited understanding of the bioavailability of food folate.

Significance of wild vegetables in micronutrient intakes of women in Vietnam: an analysis of food variety.

The association between food variety and nutrient intake/health status among rural women was tested in two agro-ecological settings in Vietnam. Special emphasis was placed on the significance of wild vegetables 'Rau Dai' in micronutrient supply and on the usefulness of food variety analysis in determining their current role. Data from 7-day food frequency interviews and a nutrition/health survey with 93 and 103 rural women in the Mekong Delta and the Central Highlands, respectively, were used in the analysis. Energy and nutrient intakes in the groups with the highest food variety score (FVS) (high = > or = 21) in the two regions were compared to those with the lowest food variety score (low = < or = 15). The high FVS groups in both regions also had a more diversified diet in terms of food categories. With the exception of low iron and riboflavin intakes in all groups, the high FVS groups had relatively adequate diets. A large variety of vegetables was used and only approximately half of the vegetable species were cultivated. In both regions the high FVS groups used a significantly greater variety of vegetables than the low FVS groups. Wild vegetables contributed significantly to the overall micronutrient intakes, mostly carotene, vitamin C and calcium intakes, but only the contribution to carotene intake was significantly higher in the high FVS group. Overall, we conclude that a food variety analysis is a useful tool in capturing the dietary role of wild vegetables.

Micronutrient composition and nutritional importance of gathered vegetables in Vietnam.

This article presents the nutrient composition and nutritional contribution of naturally occurring vegetables (NOV) in four villages in Vietnam. The frequency of consumption and quantities eaten were evaluated within the overall diet of 101 and 110 women in the Mekong Delta (flood period and rainy season, respectively) and of 103 women in the Central Highlands (rainy season). The concentrations of Ca, Fe and Zn were analysed in 28 species, and presence of tannins and phytic acid in 19 species. Provitamin carotenoids were determined in 11 species. Over 90% of the women consumed NOV, and they contributed 43, 72 and 75% (Central Highlands, Mekong rainy and Mekong flood period, respectively) of the total quantities of vegetables eaten. Aquatic species such as lpomoea aquatica and Limnocharis flava, and terrestrial vegetables such as Schismattoglottis calyptrata, were among the most commonly consumed vegetables. They were eaten in mean daily quantities exceeding 50 g and had high concentrations of Ca, Fe and beta-carotene. Other major aquatic vegetables included the stems of Eleocharis sp., Nelumbo nucifera and Nymphea lotus, all of which were used as salad vegetables and had relatively low nutrient density. The problems of assessing the current role of NOV due to lack of data on intakes in the context of the overall diets, as well as data on their composition, are addressed.

Value of traditional foods in meeting macro- and micronutrient needs: the wild plant connection.

The importance of edible wild plants may be traced to antiquity but systematic studies are recent. Anthropologists, botanists, ecologists, food scientists, geographers, nutritionists, physicians and sociologists have investigated cultural aspects and nutrient composition of edible species. Important contributions to the diet from edible wild plants are well documented and numerous studies reveal roles played by 'lesser-known' species when meeting macro- and micronutrient needs of groups at risk, whether infants and children, pregnant and/or lactating women, or the elderly. The literature is vast and scattered but information on the macro- and micronutrient content of wild plants and their importance to the human diet appear in five kinds of publications: cultural works by social scientists, descriptions and inventories by botanists, dietary assessment studies by nutritionists, intervention programmes managed by epidemiologists and physicians, and composition data generally conducted by food scientists and chemists. Many macro- and micronutrient-dense wild species deserve greater attention but lack of adequate nutrient databases, whether by region or nation, limit educational efforts to improve diets in many Third World areas. Limited and uneven compositional data generally reflect factors of cost and personal interest in key nutrients. Whilst edible wild plants are regularly deprecated by policy makers and considered to be the 'weeds of agriculture', it would be tragic if this led to loss of ability to identify and consume these important available species.

The role of vascular smooth muscle cell integrins in the compaction and mechanical strengthening of a tissue-engineered blood vessel.

Vascular smooth muscle cells (VSMC) influence vessel structure and function during normal development, and in disease states. VSMC interactions with extracellular matrix, via cell surface integrins, play an important role in these processes. A greater understanding of the molecular basis of these interactions is also critical to advances in the field of cardiovascular tissue engineering. This study examined the role of VSMC integrins in the spontaneous compaction and eventual strengthening of a rudimentary tissue-engineered blood vessel (TEBV) consisting of a fibrillar type I collagen network populated by human aortic smooth muscle cells. Using integrin subunit-specific antibodies, we demonstrated that anti-beta1 (Mab13 and P4C10) and anti-alpha2 (P1E6) antibodies that inhibit aortic smooth muscle cell (AoSMC) adhesion to collagen, also significantly inhibit TEBV compaction during the 24-hour period following TEBV construction. However, no difference in the tensile stress of antibody-treated and control TEBVs was observed at this time point. In contrast, 72 hours after construction, the inhibitory effect of anti-integrin antibodies on compaction had been overcome but tensile stress was decreased in TEBVs treated with anti-alpha2/anti-beta1 antibodies when compared to controls. These data provide evidence linking VSMC integrins, specifically the alpha2beta1 integrin, with the initial compaction, as well as, the postcompaction strengthening of the TEBV.