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Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study, (3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0), G(1), and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs. View Publication

The functional characteristics were compared for acute myelogenous leukemia (AML) cells (native blasts and AML cell lines) cultured in three serum-free media (X-vivo 10, X-vivo 15, [Bio-Whitacker, Walkersville, MD] and StemSpan [Stem Cell Technologies, Vancouver, BC, Canada]) and in medium containing 10% inactivated fetal calf serum (FCS). For native AML blasts the following functions were compared: (1) autonomous and cytokine-dependent proliferation; (2) frequency of clonogenic cell; and (3) constitutive cytokine secretion. AML blast proliferation differed between patients independent of the culture medium used, and clonogenic cells were maintained after in vitro culture in all media. In contrast, constitutive cytokine secretion was higher for cells cultured in StemSpan and FCS-containing medium than for cells cultured in the X-vivo media. Native AML blasts incubated in StemSpan also showed a low frequency of apoptotic cells. The three serum-free media could also be used for long-term expansion of well-characterized AML cell lines, but the optimal medium for cell expansion and cytokine secretion differed between cell lines. We conclude that standardized serum-free culture conditions can be used for in vitro studies of native AML blasts and AML cell lines.

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

Malignant gliomas are the most common and deadly primary malignant brain tumors. In vivo orthotopic models could doubtless represent an appropriate tool to test novel treatment for gliomas. However, methods commonly used to monitor the growth of glioma inside the mouse brain are time consuming and invasive. We tested the reliability of a minimally invasive procedure, based on a secreted luciferase (Gaussia luciferase), to frequently monitor the changes of glioma size. Gluc activity was evaluated from blood samples collected from the tail tip of mice twice a week, allowing to make a growth curve for the tumors. We validated the correlation between Gluc activity and tumor size by analysing the tumor after brain dissection. We found that this method is reliable for monitoring human glioma transplanted in immunodeficient mice, but it has strong limitation in immunocompetent models, where an immune response against the luciferase is developed during the first weeks after transplant.

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine, TPA, IBMX/forskolin), will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study, we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover, the induced TH present in these cells was enzymatically active, resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover, hNTs may serve, in vivo, as a source of human DA neurons for use in transplantation therapies.

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study, the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also, however, CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies, mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes, it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes, the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here, we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFN$\gamma$/TNF-$\alpha$+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN, whereas without oil, antigen is distributed in the medullary region. Following oil immunization, CXCL10-producing inflammatory monocytes accumulate in the IFR, which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment, CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy.

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (textless 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

Sodium butyrate, at millimolar concentrations, when added to cell cultures produces many morphological and biochemical modifications in a reversible manner. Some of them occur in all cell lines. They concern regulatory mechanisms of gene expression and cell growth: an hyperacetylation of histone resulting from an inhibition of histone deacetylase and an arrest of cell proliferation are almost constantly observed. Some other modifications vary from one cell type to another: induction of proteins, including enzymes, hormones, hemoglobin, inhibition of cell differentiation, reversion of transformed characteristics of cells to normal morphological and biochemical pattern, increase in interferon antiviral efficiency and induction of integrated viruses. Most if not all these effects of butyrate could result from histone hyperacetylation, from changes in chromatin structures as measured by accessibility to DNases and from modifications in cytoskeleton assembly. We do not know at the present time whether butyrate acts on a very specific target site in cell or if it acts on several cell components.

Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product, P-glycoprotein, and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis, with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-), respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However, recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia, but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally, the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors, makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings, it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers.