In recent years, national reports have called for undergraduate laboratory education that engages students in authentic research experiences. As a result, a number of course-based undergraduate research experiences (CUREs) have been developed in biological sciences and some specifically in microbiology. Students benefit from CUREs much like in traditional mentored research experiences, where students carry out independent projects in faculty laboratories. These benefits include increased self-efficacy in research skills, enhanced identification as scientists, and higher graduation rates in science, technology, engineering, and mathematics majors. Because mentored research experiences are not readily available to every student, CUREs represent a potential mechanism to democratize the research experience by providing such opportunities to all students. However, many of existing CUREs described in the literature are designed for advanced undergraduates or are limited to a small number of students. Here, we report student outcomes from a large-enrollment introductory CURE on soil microbiomes that engages students in a real-world context with microbiology. In pre- and post-course surveys, students reported significant gains in self-efficacy on a number of research skills. These results are triangulated with post-course survey data on project ownership, sense of community, and CURE design elements such as collaboration, iteration, discovery, and relevance.

Purpose of Review: Describe the rationale for preconditioning MSCs prior to use as therapy and the state-of-the-art of using preconditioning of MSCs in clinical settings. Recent Findings: Mounting preclinical data supports preconditioning of mesenchymal stromal cells (MSCs) to enhance their therapeutic efficacy. Most research has focused on cytokine priming and hypoxic preconditioning, while other approaches, such as glycoengineering, remain relatively understudied. Despite strong preclinical data, clinical evidence supporting preconditioning strategies are limited to six Phase I clinical trials (most of them in progress). Summary: Here, we succinctly discuss the rationale for preconditioning using cytokines, hypoxia, and glycoengineering, while elaborating on the respective clinical experiences. Overall, we note that preconditioning is highly dependent on the desired application, and therefore requires elucidating the mechanism of action of the MSCs used for therapy. Preconditioning may also help mitigate heterogeneity of MSC lots. Based on the remarkable safety profile of MSCs, even when used in allogeneic settings, the role of preconditioning prior to their final formulation might be the key to reach expected therapeutic outcomes.

Real-time neutron imaging was utilized to produce a movie-like series of radiographs for in-situ observation of the remixing of liquid state immiscibility that occurs in equiatomic CoCrCu with the addition of Ni. A previous neutron imaging study demonstrated that liquid state immiscibility can be observed in-situ for the equiatomic CoCrCu alloy. In this follow-up study, equiatomic buttons of CoCrCu were placed alongside small Ni buttons inside an alumina crucible in a high-temperature vacuum furnace. The mass of the Ni buttons was specifically selected such that when melted in the same crucible as the CoCrCu buttons, the overall composition would become equiatomic CoCrCuNi. Neutron imaging was simultaneously carried out to capture 10 radiographs in 20 °C steps from 1000 °C to 1500 °C and back down to 1000 °C. This, in turn, produced a movie-like series of radiographs that allow for the observation of the buttons melting, the transition from immiscible to miscible as Ni is alloyed into the CoCrCu system, and solidification. This novel imaging process showed the phase-separated liquids remixing into a single-phase liquid when Ni dissolves into the melt, which makes this technique crucial for understanding the liquid state behavior of these complex alloy systems. As metals are not transparent to X-ray imaging techniques at this scale, neutron imaging of melting and solidification allows for the observation of liquid state phase changes in real time. Thermodynamic calculations of the isopleth for CoCrCuNix were carried out to compare the observed results to the predictions resulting from the current Thermo-Calc TCHEA3 thermodynamic database. The calculations show a very good agreement with the experimental results, as the calculations indicate that the CoCrCuNix alloy solidifies from a single-phase liquid when x ≥ 0.275, which is close to the nominal concentration of the CoCrCuNi alloy (x = 0.25). The neutron imaging shows that the solidification of CoCrCuNi results from a single-phase liquid. This is evident as no changes in the neutron attenuation were observed during the solidification of the CoCrCuNi alloy.

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.

Patients with Snyder-Robinson Syndrome (SRS) exhibit deficient Spermidine Synthase (SMS) gene expression, which causes neurodevelopmental defects and osteoporosis, often leading to extremely fragile bones. To determine the underlying mechanism for impaired bone formation, we modelled the disease by silencing SMS in human bone marrow - derived multipotent stromal cells (MSCs) derived from healthy donors. We found that silencing SMS in MSCs led to reduced cell proliferation and deficient bone formation in vitro, as evidenced by reduced mineralization and decreased bone sialoprotein expression. Furthermore, transplantation of MSCs in osteoconductive scaffolds into immune deficient mice shows that silencing SMS also reduces ectopic bone formation in vivo. Tag-Seq Gene Expression Profiling shows that deficient SMS expression causes strong transcriptome changes, especially in genes related to cell proliferation and metabolic functions. Similarly, metabolome analysis by mass spectrometry, shows that silencing SMS strongly impacts glucose metabolism. This was consistent with observations using electron microscopy, where SMS deficient MSCs show high levels of mitochondrial fusion. In line with these findings, SMS deficiency causes a reduction in glucose consumption and increase in lactate secretion. Our data also suggests that SMS deficiency affects iron metabolism in the cells, which we hypothesize is linked to deficient mitochondrial function. Altogether, our studies suggest that SMS deficiency causes strong transcriptomic and metabolic changes in MSCs, which are likely associated with the observed impaired osteogenesis both in vitro and in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.

INTRODUCTION: The etiology and progression of sporadic Alzheimer's Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (Aβ) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in Aβ clearance, and alterations in microglial function due to aging or disease may result in Aβ accumulation and deleterious effects on neuronal function. However, studying these complex factors in vivo , where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and Aβ clearance) and proteomic response to exogenous Aβ. METHODS: We established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days in vitro (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled Aβ (FITC-Aβ) particles for varying durations. Images were analyzed to determine the number of FITC-Aβ particles after specific lengths of exposure. A group of cells were stained for βIII-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio). RESULTS AND DISCUSSION: FITC-Aβ particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aβ particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-Aβ significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-Aβ addition revealed a significant increase in proinflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6) in tri-culture, but not co-culture. In addition, Aβ addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and Aβ clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.

For hundreds of indications, mesenchymal stromal cells (MSCs) have not achieved the expected therapeutic efficacy due to an inability of the cells to reach target tissues. We show that inducing high mannose N-glycans either chemically, using the mannosidase I inhibitor Kifunensine, or genetically, using an shRNA to silence the expression of mannosidase I A1 (MAN1A1), strongly increases the motility of MSCs. We show that treatment of MSCs with Kifunensine increases cell migration toward bone fracture sites after percutaneous injection, and toward lungs after intravenous injection. Mechanistically, high mannose N-glycans reduce the contact area of cells with its substrate. Silencing MAN1A1 also makes cells softer, suggesting that an increase of high mannose N-glycoforms may change the physical properties of the cell membrane. To determine if treatment with Kifunensine is feasible for future clinical studies, we used mass spectrometry to analyze the N-glycan profile of MSCs over time and demonstrate that the effect of Kifunensine is both transitory and at the expense of specific N-glycoforms, including fucosylations. Finally, we also investigated the effect of Kifunensine on cell proliferation, differentiation, and the secretion profile of MSCs. Our results support the notion of inducing high mannose N-glycans in MSCs in order to enhance their migration potential.