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Metformin is an antidiabetic drug, increasingly prescribed in pregnancy and has been shown to cross the human placenta. The mechanisms underlying placental metformin transfer remain unclear. This study investigated the roles of drug transporters and paracellular diffusion in the bidirectional transfer of metformin across the human placental syncytiotrophoblast using placental perfusion experiments and computational modelling. 14C-metformin transfer was observed in the maternal to fetal and fetal to maternal directions and was not competitively inhibited by 5 mM unlabelled metformin. Computational modelling of the data was consistent with overall placental transfer via paracellular diffusion. Interestingly, the model also predicted a transient peak in fetal 14C-metformin release due to trans-stimulation of OCT3 by unlabelled metformin at the basal membrane. To test this hypothesis a second experiment was designed. OCT3 substrates (5 mM metformin, 5 mM verapamil and 10 mM decynium-22) added to the fetal artery trans-stimulated release of 14C-metformin from the placenta into the fetal circulation, while 5 mM corticosterone did not. This study demonstrated activity of OCT3 transporters on the basal membrane of the human syncytiotrophoblast. However, we did not detect a contribution of either OCT3 or apical membrane transporters to overall materno-fetal transfer, which could be represented adequately by paracellular diffusion in our system.

The isolated perfused placental cotyledon technique has led to numerous advances in placental biology. Combining placental perfusion with mathematical modelling provides an additional level of insight into placental function. Mathematical modelling of perfusion data provides a quantitative framework to test the understanding of the underlying biology and to explore how different processes work together within the placenta as part of an integrated system. The perfusion technique provides a high degree of control over the experimental conditions as well as regular measurements of functional parameters such as pressure, solute concentrations and pH over time. This level of control is ideal for modelling as it allows placental function to be studied across a wide range of different conditions which permits robust testing of mathematical models. By placing quantitative values on different processes (e.g. transport, metabolism, blood flow), their relative contribution to the system can be estimated and those most likely to become rate-limiting identified. Using a combined placental perfusion and modelling approach, placental metabolism was shown to be a more important determinant of amino acid and fatty acid transfer. In contrast, metabolism was a less important determinant of placental cortisol transfer than initially thought. Identifying the rate-limiting factors in the system allows future work to be focused on the factors that are most likely to underlie placental dysfunction. A combined experimental and modelling approach using placental perfusions promotes an integrated view of placental physiology that can more effectively identify the processes leading to placental pathologies.

The factors determining fatty acid transfer across the placenta are not fully understood. This study used a combined experimental and computational modeling approach to explore placental transfer of nonesterified fatty acids and identify the rate-determining processes. Isolated perfused human placenta was used to study the uptake and transfer of 13C-fatty acids and the release of endogenous fatty acids. Only 6.2 ± 0.8% of the maternal 13C-fatty acids taken up by the placenta was delivered to the fetal circulation. Of the unlabeled fatty acids released from endogenous lipid pools, 78 ± 5% was recovered in the maternal circulation and 22 ± 5% in the fetal circulation. Computational modeling indicated that fatty acid metabolism was necessary to explain the discrepancy between uptake and delivery of 13C-fatty acids. Without metabolism, the model overpredicts the fetal delivery of 13C-fatty acids 15-fold. Metabolic rate was predicted to be the main determinant of uptake from the maternal circulation. The microvillous membrane had a greater fatty acid transport capacity than the basal membrane. This study suggests that incorporation of fatty acids into placental lipid pools may modulate their transfer to the fetus. Future work needs to focus on the factors regulating fatty acid incorporation into lipid pools.

The placenta mediates the transfer of maternal nutrients into the fetal circulation while removing fetal waste products, drugs and environmental toxins that may otherwise be detrimental to fetal development. This study investigated the role of drug transporters and protein binding in the transfer of the antidiabetic drug glibenclamide across the human placental syncytiotrophoblast using placental perfusion experiments and computational modeling. In the absence of albumin, placental glibenclamide uptake from the fetal circulation was not affected by competitive inhibition with bromosulphothalein (BSP), indicating that OATP2B1 does not mediate placental glibenclamide uptake from the fetus. In the presence of maternal and fetal albumin, BSP increased placental glibenclamide uptake from the fetal circulation by displacing glibenclamide from BSA, increasing the free fraction of glibenclamide driving diffusive transport. The P-gp and BCRP inhibitor GF120918 did not affect placental glibenclamide uptake from the maternal circulation and as such this study did not find any evidence for the apical efflux transporters in placental glibenclamide transfer. Computational modeling confirmed that albumin binding and not transporter activity, is the dominant factor in the transfer of glibenclamide across the human placenta. The effect of BSP binding to albumin on promoting the diffusive transfer of glibenclamide highlights the importance of drug-protein binding interactions and their interpretation using computational modeling.

Placental amino acid transport is required for fetal development and impaired transport has been associated with poor fetal growth. It is well known that placental amino acid transport is mediated by a broad array of specific membrane transporters with overlapping substrate specificity. However, it is not fully understood how these transporters function, both individually and as an integrated system. We propose that mathematical modelling could help in further elucidating the underlying mechanisms of how these transporters mediate placental amino acid transport. The aim of this work is to model the sodium independent transport of serine, which has been assumed to follow an obligatory exchange mechanism. However, previous amino acid uptake experiments in human placental microvillous plasma membrane vesicles have persistently produced results that are seemingly incompatible with such a mechanism; i.e. transport has been observed under zero-trans conditions, in the absence of internal substrates inside the vesicles to drive exchange. This observation raises two alternative hypotheses; (i) either exchange is not fully obligatory, or (ii) exchange is indeed obligatory, but an unforeseen initial concentration of amino acid substrate is present within the vesicle which could drive exchange. To investigate these possibilities, a mathematical model for tracer uptake was developed based on carrier mediated transport, which can represent either facilitated diffusion or obligatory exchange (also referred to as uniport and antiport mechanisms, respectively). In vitro measurements of serine uptake by placental microvillous membrane vesicles were carried out and the model applied to interpret the results based on the measured apparent Michaelis–Menten parameters Km and Vmax. In addition, based on model predictions, a new time series experiment was implemented to distinguish the hypothesised transporter mechanisms. Analysis of the results indicated the presence of a facilitated transport component, while based on the model no evidence for substantial levels of endogenous amino acids within the vesicle was found., Highlights • Initial rate and time course data for serine uptake in placental membrane vesicles. • Integrated model analysisof facilitative diffusion vs obligatory exchange. • Dependency apparent Michaelis–Menten constants on internal concentrations. • Uptake in placental vesicles was consistent with a facilitative transport component. • No effects of any internal endogenous substrate in vesicles were apparent.

Skeletal regeneration and tissue engineering strategies rely critically on the efficient expansion of progenitor cell populations whilst simultaneously preserving multipotentiality and the ability to induce differentiation towards bone and cartilage. Cell population heterogeneity has a significant impact on this process, but is currently poorly quantified, hampering the interpretation of experimental results and the design of optimised expansion protocols. The objective of this study was to characterise individual human bone marrow stromal cell heterogeneity in terms of colony expansion potential. For this purpose, a novel two-stage CFU-F assay was developed in which cells from primary single cell-derived colonies were detached and reseeded again at clonal density as single cells to form new secondary colonies. This clearly demonstrated how secondary colony growth potential varies markedly both between and within primary colonies. Depending on the primary colony, cells either generated small secondary colonies only, or else a wide range of colony sizes. Using computational modelling it was shown how such colony heterogeneity could arise from hierarchical progenitor cell populations and what the limits of such a population structure were in explaining the experimental data. In addition the model demonstrated the significant potential impact of cell mobility on expansion potential and its implications for inducing population heterogeneity. This combined experimental-computational approach will ascertain the impact of cell culture protocols on the expansion potential and functional composition of heterogeneous progenitor populations. Such insights are likely to be of crucial importance for the success of skeletal regeneration strategies.

s / Placenta 35 (2014) A1eA112 A99 Results: IF dams consumed 29±1% less food and gained less weight (P Ala>BCH>Pro>Gly>MeAIB>Met. Conclusion: The identity of amino acids present on the internal face of MVM dictates serine uptake capacity across MVM. The broad internal amino acid selectivity by system L in MVM indicates that both the composition and concentration of freely-exchangeable amino acids within syncytiotrophoblast is likely to regulate system L exchange capacity in MVM. Collectively these observations provide further experimental support for previous model predictions regarding serine uptake by system L exchange transporters. P2.123-N. STATUS ANALYSIS OF TUMOUR ASSOCIATED FACTORS IN HUMAN PLACENTAE AND STEM-LIKE CELLS DERIVED FROM PLACENTAL CELL LINES Rham Balahmar, Shiva Sivasubramaniam Nottingham Trent university

Introduction: The Na+-independent uptake of serine into microvillous plasma membrane (MVM) vesicles is thought to be mediated by the exchange transporter LAT2. Exchangers are predicted to transport one amino acid (aa) in exchange for another in an obligatory 1:1 manner. However, the observation that serine is transported across MVM under zero-trans conditions, in the absence of internal exchangeable aa, questions whether exchange is indeed obligatory. We revisit the exchange-for-nothing phenomenon by developing mathematical models to examine obligate versus non-obligate exchange, by introducing a hypothesised facilitated component. Methods: Mathematical models were developed based on carrier-mediated transport via a) obligatory exchange, or b) non-obligatory exchange, corresponding to facilitative diffusion. Models were tested by measuring uptake of 7.5 �M 14C-serine into MVM vesicles isolated from normal term placentas, preloaded with either 0 �M or 250 �M (equal to extravesicular serine concentration) or 1 mM serine (outwardly directed substrate gradient to drive facilitated transport). Results: Trans-stimulation of tracer uptake was observed with increasing intra-vesicular serine concentration indicating an exchange component. However, if exchange were obligatory, the model predicted that equilibrium values would be reflected by ratios of intra- and extra-vesicular concentrations, which was not observed. Crucially, the model predicted that for a facilitated transport component, an outwardly directed aa gradient would cause an overshoot of tracer uptake, which was indeed observed.Conclusion: Models were consistent with serine uptake into MVM vesicles by non-obligatory exchange, or exchange together with an as yet uncharacterised facilitated transporter in parallel. This study emphasizes the utility of mathematical modelling in developing an improved quantitative understanding of aa transporter mechanisms. The physiological role for a facilitated transport component on the MVM is at present unclear and warrants further investigation.