ASU Scholarship Showcase

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population that is capable of nearly indefinite expansion and subsequent differentiation into the various neuronal and supporting cell types that comprise the CNS. However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS. In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation. Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures. The ability to manipulate WNT signaling to generate regionally specific NPCs and neurons will be useful for studying human neural development and will greatly enhance the translational potential of hPSCs for neural-related therapies.

Although the majority of late-onset Alzheimer's disease (AD) patients are labeled sporadic, multiple genetic risk variants have been identified, the most powerful and prevalent of which is the e4 variant of the Apolipoprotein E (APOE) gene. Here, we generated human induced pluripotent stem cell (hiPSC) lines from the peripheral blood mononuclear cells (PBMCs) of a clinically diagnosed AD patient [ASUi003-A] and a non-demented control (NDC) patient [ASUi004-A] homozygous for the APOE4 risk allele. These hiPSCs maintained their original genotype, expressed pluripotency markers, exhibited a normal karyotype, and retained the ability to differentiate into cells representative of the three germ layers.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

Adult and pluripotent stem cells represent a ready supply of cellular raw materials that can be used to generate the functionally mature cells needed to replace damaged or diseased heart tissue. However, the use of stem cells for cardiac regenerative therapies is limited by the low efficiency by which stem cells are differentiated in vitro to cardiac lineages as well as the inability to effectively deliver stem cells and their derivatives to regions of damaged myocardium. In this review, we discuss the various biomaterial-based approaches that are being implemented to direct stem cell fate both in vitro and in vivo. First, we discuss the stem cell types available for cardiac repair and the engineering of naturally and synthetically derived biomaterials to direct their in vitro differentiation to the cell types that comprise heart tissue. Next, we describe biomaterial-based approaches that are being implemented to enhance the in vivo integration and differentiation of stem cells delivered to areas of cardiac damage. Finally, we present emerging trends of using stem cell-based biomaterial approaches to deliver pro-survival factors and fully vascularized tissue to the damaged and diseased cardiac tissue.

Outer membrane vesicles (OMVs) isolated from Salmonella Typhimurium are potentially useful for developing subunit vaccines because of high immunogenicity and protective efficacy. However, flagella might remain in OMV pellets following OMV purification, resulting in non-essential immune responses and counteraction of bacterial protective immune responses when developing a vaccine against infection of multiple serotypes Salmonella. In this study, a flagellin-deficient S. Typhimurium mutant was constructed. Lipopolysaccharide profiles, protein profiles and cryo-electron microscopy revealed that there were no significant differences between the wild-type and mutant OMVs, with the exception of a large amount of flagellin in the wild-type OMVs. Neither the wild-type OMVs nor the non-flagellin OMVs were toxic to macrophages. Mice immunized with the non-flagellin OMVs produced high concentrations of IgG. The non-flagellin OMVs elicited strong mucosal antibody responses in mice when administered via the intranasal route in addition to provoking higher cross-reactive immune responses against OMPs isolated from S. Choleraesuis and S. Enteritidis. Both intranasal and intraperitoneal immunization with the non-flagellin OMVs provided efficient protection against heterologous S. Choleraesuis and S. Enteritidis challenge. Our results indicate that the flagellin-deficient OMVs may represent a new vaccine platform that could be exploited to facilitate the production of a broadly protective vaccine.

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 10⁷ 50% tissue culture infective doses (TCID₅₀)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.

Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c⁺ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.