Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development, modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (B70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (4250%) MN production in chemically defined adherent cultures. Furthermore, we show that Islet-1 is essential for formation of mature and functional human MNs, but, unlike its mouse counterpart, does not regulate cell survival or suppress the V2a interneuron fate. Together, our discoveries improve the strategy for MN derivation, advance our understanding of human neural specification and MN development, and provide invaluable tools for human developmental studies, drug discovery and regenerative medicine.

The visual selection of specific cells within an ex vivo brain slice, combined with whole-cell patch clamp recording and capillary electrophoresis (CE)–mass spectrometry (MS)-based metabolomics, yields high chemical information on the selected cells. By providing access to a cell’s intracellular environment, the whole-cell patch clamp technique allows one to record the cell’s physiological activity. A patch clamp pipet is used to withdraw ∼3 pL of cytoplasm for metabolomic analysis using CE–MS. Sampling the cytoplasm, rather than an intact isolated neuron, ensures that the sample arises from the cell of interest and that structures such as presynaptic terminals from surrounding, nontargeted neurons are not sampled. We sampled the rat thalamus, a well-defined system containing gamma-aminobutyric acid (GABA)-ergic and glutamatergic neurons. The approach was validated by recording and sampling from glutamatergic thalamocortical neurons, which receive major synaptic input from GABAergic thalamic reticular nucleus neurons, as well as neurons and astrocytes from the ventral basal nucleus and the dorsal lateral geniculate nucleus. From the analysis of the cytoplasm of glutamatergic cells, approximately 60 metabolites were detected, none of which corresponded to the compound GABA. However, GABA was successfully detected when sampling the cytoplasm of GABAergic neurons, demonstrating the exclusive nature of our cytoplasmic sampling approach. The combination of whole-cell patch clamp with single cell cytoplasm metabolomics provides the ability to link the physiological activity of neurons and astrocytes with their neurochemical state. The observed differences in the metabolome of these neurons underscore the striking cell to cell heterogeneity in the brain.

Objectives Healthy pregnancy diet is important for infant development, but maternal dietary effects on establishment of the infant gut microbiome are not well understood. Our goal was to test the feasibility of conducting a randomized controlled trial (RCT) to implement a diet intervention and collect survey data and biospecimens from pregnant women and their infants. Methods Employing a 2-arm RCT, we recruited women (n = 27) in mid-pregnancy from a prenatal care clinic, randomized participants to the intervention (n = 13) or to usual care (n = 14), and followed to 6 wks postpartum. Data collection included surveys, maternal blood, urine, and stool at three time points (T1 = 25 and T2 = 36 wks gestation [pre- and 4 wks post-intervention initiation, respectively]; T3 = 6 wks postpartum), and infant stool at 6 wks. The intervention was initiated at 32 wks gestation and continued until birth of the baby. In wk 1, participants received non-perishable high fiber foods (whole wheat cereal, oatmeal, dried fruit, canned beans) as well as olive oil, vinegar, recipes for salad dressing and side dishes, and general nutrition information. Partnering with a hospital catering service, the intervention included weekly food delivery of 3 large prepared salads, 2 quarts of soup including either legumes or whole grains (e.g., beans, barley), and 5 pieces of fresh fruit (e.g., apples, oranges). Results Our sample is considered entirely rural and maternal characteristics reflected the local population: 100% White, 17% Hispanic or Native American; 89% at least some college; 26% Medicaid; 33% ever smoked. Mean maternal age was 29.6 y (range 20–40 y), mean pre-pregnancy BMI was 26.9 kg/m2 (range 18.5-41.6). Data collection adherence was high with few missing data points: T1 = 100% for survey, blood, urine, stool; T2 = 93% for stool, 96% for blood and urine; T3 = 93% for survey, blood, urine, and stool). Overall satisfaction was high with 85% reporting satisfied or very satisfied and important qualitative insights were gained from participants. Conclusions This pilot trial produced valuable information to effectively refine the intervention so that it can be tested in a larger, longer study using a factorial design to test the effects of pregnancy diet and/or postpartum diet of breastfeeding moms on the establishment of the infant microbiome. Funding Sources MSU internal funds (AgBioResearch and faculty start-up).

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Na,K‐ATPase (NKA) is an integral membrane protein that plays a central role in ionic homeostasis in animals by mediating transport of Na+ and K+ ions against their electrochemical gradients. NKA is a heterodimer of α and β subunits. It is not yet clear if the native structure of NKA consists of αβ‐protomers or higher order oligomeric states. α‐ α associations were earlier demonstrated using in vitro pull down assays with isolated ATP‐binding domain (ABD) and full‐length α (Costa et al., 2002). Our objective is to elucidate the oligomeric state of NKA throughout maturation and trafficking and the role it plays in protein‐protein interactions. Consistent with previous observations, results from our co‐immunoprecipitation experiments using lysates from HEK 293 cells stably expressing α‐subunit with v5‐ or myc‐tagged NKA α‐subunits demonstrate α‐α interaction. Moreover, this self‐association is stabilized by including 2mM Mg‐ATP in the assay. Our preliminary results from confocal imaging of HEK 293 cells co‐expressing GFP‐tagged ABD and mcherry‐tagged full length α‐subunit revealed GFP‐ABD in the plasma membrane (PM), consistent with the isolated ABD being “carried” to the PM with full‐length α‐subunits. We also found that the α‐subunit lacking the M4M5 cytoplasmic loop (αΔ45LL), and thus unable to bind ATP, was retained in ER. Studies are underway to see if expressing the isolated ABD will rescue αΔ45LL and facilitate its delivery to the PM. This work was supported by NIH grant GM061583.