Apical-out airway organoids as a platform for studying viral infections and screening for antiviral drugs.

Airway organoids are polarized 3D epithelial structures that recapitulate the organization and many of the key functions of the in vivo tissue. They present an attractive model that can overcome some of the limitations of traditional 2D and Air-Liquid Interface (ALI) models, yet the limited accessibility of the organoids' apical side has hindered their applications in studies focusing on host-pathogen interactions. Here, we describe a scalable, fast and efficient way to generate airway organoids with the apical side externally exposed.

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side.

Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells.

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential.

Targeting primitive chronic myeloid leukemia cells by effective inhibition of a new AHI-1-BCR-ABL-JAK2 complex.

Background: Imatinib mesylate (IM) induces clinical remission of chronic myeloid leukemia (CML). The Abelson helper integration site 1 (AHI-1) oncoprotein interacts with BCR-ABL and Janus kinase 2 (JAK2) to mediate IM response of primitive CML cells, but the effect of the interaction complex on the response to ABL and JAK2 inhibitors is unknown.

Methods: The AHI-1-BCR-ABL-JAK2 interaction complex was analyzed by mutational analysis and coimmunoprecipitation.

Properties of CD34+ CML stem/progenitor cells that correlate with different clinical responses to imatinib mesylate.

Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients, we now demonstrate that some, but not all, of these parameters correlate with subsequent clinical response to IM therapy.

Reduction in multi-lineage and erythroid progenitors distinguishes myelodysplastic syndromes from non-malignant cytopenias.

We studied the diagnostic role of CFC assays in myelodysplastic syndromes (MDS) using CFC data from bone marrow (BM) and peripheral blood (PB) of 221 MDS patients, 51 patients with non-malignant causes of cytopenia and/or dysplasia and 50 normal controls. A consistent decrease in BM but not PB multi-lineage and erythroid progenitor frequencies was seen in patients with MDS compared to controls (P<0.05).

High-resolution whole genome tiling path array CGH analysis of CD34+ cells from patients with low-risk myelodysplastic syndromes reveals cryptic copy number alterations and predicts overall and leukemia-free survival.

Myelodysplastic syndromes (MDSs) pose an important diagnostic and treatment challenge because of the genetic heterogeneity and poorly understood biology of the disease. To investigate initiating genomic alterations and the potential prognostic significance of cryptic genomic changes in low-risk MDS, we performed whole genome tiling path array comparative genomic hybridization (aCGH) on CD34(+) cells from 44 patients with an International Prognostic Scoring System score less than or equal to 1.0.

Stem cell biomarkers in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal multi-step myeloproliferative disease that is initially produced and ultimately sustained by a rare subpopulation of BCR-ABL+ cells with multi-lineage stem cell properties. These BCR-ABL+ CML stem cells are phenotypically similar to normal hematopoietic stem cells which are also maintained throughout the course of the disease at varying levels in different patients. Defining the unique properties of the leukemic stem cells that produce the chronic phase of CML has therefore had to rely heavily on access to samples from rare patients in which the stem cell compartment is dominated by leukemic elements.

Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation.

Improved purification of hematopoietic stem cells based on their elevated aldehyde dehydrogenase activity.

Background And Objectives: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo.

Design And Methods: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers.

Instability of BCR-ABL gene in primary and cultured chronic myeloid leukemia stem cells.

Background: Imatinib mesylate treatment causes remissions in a majority of patients with chronic myeloid leukemia (CML), but relapses are an increasing problem. We hypothesized that imatinib-resistant leukemic cells emerge from CML stem cells that acquire BCR-ABL gene mutations even before exposure to BCR-ABL-targeted agents such as imatinib.

Methods: Lineage-negative (i.

The challenges of targeting chronic myeloid leukemia stem cells.

Chronic myeloid leukemia (CML) is sustained by a clonally amplified population of Bcr Abl-positive pluripotent stem cells. Persistence of a large, functionally intact yet suppressed residual normal hematopoietic stem cell population in most patients with CML has made it possible to aim at the development of curative therapies. However, achieving this goal requires the identification of agents that will eradicate the leukemic stem cell population.

Intermittent exposure of primitive quiescent chronic myeloid leukemia cells to granulocyte-colony stimulating factor in vitro promotes their elimination by imatinib mesylate.

Purpose: Primitive quiescent chronic myeloid leukemia (CML) cells are biologically resistant to imatinib mesylate, an inhibitor of the p210(BCR-ABL) kinase. The present study was designed to investigate whether either continuous or intermittent exposure of these cells to granulocyte-colony stimulating factor (G-CSF) in vitro can overcome this limitation to the effectiveness of imatinib mesylate therapy.

Experimental Design: CD34(+) leukemic cells were isolated from six newly diagnosed chronic phase CML patients and cultured for 12 days in serum-free medium with or without G-CSF and/or imatinib mesylate present either continuously or intermittently (three cycles of G-CSF for 0, 1, or 4 days +/- imatinib mesylate for 0, 3, or 4 days).

Isolation of subsets of immune cells.

Subsets of immune cells can be isolated before analysis by the enzyme-linked immunospot (ELISPOT) assay with various cell separation techniques. This chapter describes techniques to select desired cells or deplete unwanted cells by crosslinking cells to dense or magnetic particles for subsequent separation. The RosetteSep method can be used to isolate specific cell types directly from human whole blood, using the red blood cells (RBCs) present in the sample as dense particles.

Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome.

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies, but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice, whereas most MDS samples produced predominantly granulopoietic cells.

Deregulated expression in Ph+ human leukemias of AHI-1, a gene activated by insertional mutagenesis in mouse models of leukemia.

Ahi-1/AHI-1 (Abelson helper integration site-1) encodes a family of protein isoforms containing one Src homology 3 (SH3) domain and multiple tryptophan-aspartic acid 40 (WD40)-repeat domains. The function of these proteins is unknown, but involvement in leukemogenesis has been suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias. Here we show that in both mice and humans, Ahi-1/AHI-1 expression is highest in the most primitive hematopoietic cells with specific patterns of down-regulation in different lineages.

Identification and isolation of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs, their identification and enumeration depends on functional long-term, multilineage, in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal.

Evidence for a positive role of SHIP in the BCR-ABL-mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia.

Previous studies suggested that the SH2-containing inositol-5-phosphatase (SHIP) may play a tumor suppressor-like function in BCR-ABL-mediated leukemogenesis. To investigate this possibility, we first developed a new assay for quantitating transplantable multilineage leukemia-initiating cells (L-ICs) in hematopoietic stem cell (HSC)-enriched mouse bone marrow (BM) cells transduced with a BCR-ABL-GFP (green fluorescent protein) retrovirus. The frequency of L-ICs (1 of 430 Sca-1+lin- cells) was 7-fold lower than the frequency of HSCs in the Sca-1+lin- subset transduced with a control virus (1 of 65 cells).

New models to investigate mechanisms of disease genesis from primitive BCR-ABL(+) hematopoietic cells.

Three years ago we described a novel autocrine IL-3/G-CSF mechanism active in the leukemic CD34(+) cells from chronic myeloid leukemia (CML) patients in chronic phase (PNAS 96: 12804-12809, [1999]). We also showed that exposure of the most primitive CD34(+) cells from normal human bone marrow to excess IL-3 stimulates not only the division of these cells but also their differentiation. In contrast, both IL-3 and G-CSF cause an expansion of the more mature types of normal CD34(+) progenitors.

Primitive interleukin 3 null hematopoietic cells transduced with BCR-ABL show accelerated loss after culture of factor-independence in vitro and leukemogenic activity in vivo.

Primitive chronic myeloid leukemia cells display a unique autocrine interleukin 3 (IL-3)/granulocyte-colony-stimluating factor (G-CSF) mechanism that may explain their abnormal proliferation and differentiation control. Here we show that BCR-ABL transduction of primitive Sca-1(+) lin(-) mouse bone marrow (BM) cells causes immediate activation of IL-3, G-CSF, and granulocyte macrophage-colony-stimulating factor (GM-CSF) expression in these cells. Their autocrine IL-3-mediated growth dependence is thus demonstrable only in clonal cultures where paracrine effects are reduced.

Modulation of p210(BCR-ABL) activity in transduced primary human hematopoietic cells controls lineage programming.

Retroviral transduction of primary hematopoietic cells with human oncogenes provides a powerful approach to investigating the molecular mechanisms controlling the normal proliferation and differentiation of these cells. Here we show that primitive human CD34(+) cord blood cells, including multipotent as well as granulopoietic- and erythroid-restricted progenitors, can be efficiently transduced with a MSCV-BCR-ABL-IRES-GFP retrovirus, resulting in the sustained expression by their progeny of very high levels of tyrosine phosphorylated p210(BCR-ABL). Interestingly, even in the presence of growth factors that supported the exclusive production of granulopoietic cells from green fluorescent protein (GFP)-transduced control cells, BCR-ABL-transduced progenitor subpopulations generated large numbers of erythropoietin-independent terminally differentiating erythroid cells and reduced numbers of granulopoietic cells.

Stromal-derived factor 1 inhibits the cycling of very primitive human hematopoietic cells in vitro and in NOD/SCID mice.

Stromal-derived factor 1 (SDF-1) is a -CXC- chemokine that plays a critical role in embryonic and adult hematopoiesis, and its specific receptor, CXCR4, has been implicated in stem cell homing. In this study, it is shown that the addition of SDF-1 to long-term cultures (LTCs) of normal human marrow can selectively, reversibly, and specifically block the S-phase entry of primitive quiescent erythroid and granulopoietic colony-forming cells (CFCs) present in the adherent layer. Conversely, addition of anti-SDF-1 antibody or SDF-1(G2), a specific CXCR4 antagonist, to preactivated human LTCs prevented both types of primitive CFCs from re-entering a quiescent state, demonstrating that endogenous SDF-1 contributes to the control of primitive CFC proliferation in the LTC system.