The homeodomain gene Pitx2 is expressed in primitive hematopoietic stem/progenitor cells but not in their differentiated progeny.

OBJECTIVE: Hematopoietic stem cells (HSCs) represent a rare and incompletely characterized fraction of marrow cells that are capable of both self-renewal and differentiation into all of the mature cells in the peripheral blood. We undertook to identify genes expressed preferentially by HSCs as an initial step toward better understanding the molecular mechanisms that underlie HSC behavior. METHODS: We modified the representational difference analysis technique to isolate gene fragments present in amplified cDNA prepared from highly purified murine hematopoietic stem/progenitor cells (Lin(-)/Hoechst(low)/rhodamine(low)) and absent (or much less abundant) in amplified cDNA prepared from lineage-committed marrow cells. We went on to use one potentially important gene fragment that we isolated in this way, to screen a cDNA library prepared from these cells and to characterize the pattern of expression of the gene in hematopoietic and other cells. RESULTS: We isolated a fragment of the homeobox transcription factor Pitx2 from amplified cDNA prepared from murine hematopoietic stem/progenitor cells. From a cDNA library prepared from these cells, a full-length cDNA was isolated that corresponds to one of the three known isoforms of Pitx2 (Pitx2c). Pitx2c is expressed in murine embryonic stem (ES) cells and in hematopoietic stem/progenitor cells but not in more differentiated hematopoietic cells or in a large panel of established murine hematopoietic cell lines. Pitx2c expression was not detected after 48 hours of in vitro cytokine stimulation of hematopoietic stem/progenitor cells. CONCLUSIONS: Pitx2c is expressed in hematopoietic stem/progenitor cells but not in their differentiated progeny. The pattern of expression of Pitx2c in primitive hematopoietic stem/progenitor cells suggests that it may play a role in hematopoietic stem-cell biology.

Characterization of neurosphere cell phenotypes by flow cytometry.

BACKGROUND: Neural stem cell research regularly utilizes neurosphere cultures as a continuous source of primitive neural cells. Results from current progenitor cell assays show that these cultures contain a low number of neural progenitors. Our goal is to characterize neurosphere cultures and define subpopulations in order to purify neural progenitor cells. METHODS: Cells from embryonic mouse neurosphere cultures were stained with Hoechst 33342 and analyzed by flow cytometry. Subpopulations were sorted based on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 days in culture. RESULTS: Neurosphere cultures contain a relatively high number of cells that stain weakly with Hoechst 33342. This subpopulation is present when cultured as an entire batch in the presence of epidermal growth factor (EGF). When cultured separately, this subpopulation gives rise to a neurosphere population with essentially the same characteristics as freshly isolated embryonic mouse brain cells but contains substantially fewer weakly Hoechst-stained cells. CONCLUSIONS: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells.

Correlates between hematopoiesis and neuropoiesis: neural stem cells.

There are many parallels between the neuropoietic and lymphohematopoietic systems. The lymphohematopoietic stem/progenitor cell system has been extensively characterized, but there are still major questions relating to the definitive stem cell assay, the structure of the system (i.e., hierarchical versus cell cycle-based), and the nature of differentiation (i.e., stochastic versus deterministic). Recent data have established the existence of an epidermal growth factor (EGF)-responsive neural stem cell in adult mice. We have studied these neural progenitor/stem cells in fetal (day 15) and 2-day postnatal mice and established a single-cell progenitor assay and a variety of putative uni-, bi-, and tripotential stem cells that form in response to EGF. Neurospheres are the EGF-responsive neural units that grow in liquid culture, and we have found that cells derived from these neurospheres express a wide array of cytokines and their receptors. This will provide a window on the hemopoietic progenitor system analogous to that created by the description of in vitro growth of clonal hematopoietic progenitors.

In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions.

Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron-glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for beta-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10-11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15-18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that > 62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain.

In situ detection of individual transplanted bone marrow cells using FISH on sections of paraffin-embedded whole murine femurs.

Studies of transplantation biology rely on the detection of donor hemopoietic cells in transplant recipients. Traditionally this has been achieved through ex vivo techniques, including flow cytometric analysis of cell surface markers to detect cells expressing specific epitopes, histochemical detection of cytoplasmic proteins, and the detection of Y chromosome-specific sequences by DNA hybridization. Studies using congenic models, such as the Ly5.1/5.2 mouse, or the utilization of fluorescent dyes, such as PKH-26, have allowed more in-depth analysis of transplantation, beginning to address key issues such as cell homing through cell tracking and elucidation of the "stem cell niche." However, these methods are limited by labeling sensitivity, specificity, crossreactivity and, in the case of PKH-26 labeling, the number of cell divisions the transplanted cells can make before the signal disappears. We have developed a fluorescent in situ hybridization (FISH) technique that utilizes a murine Y chromosome-specific "painting" probe to identify in situ individual transplanted male cells in paraffin-embedded sections of female whole bone marrow while maintaining good morphological integrity. This method is highly sensitive and specific, labeling more than 99% of male cells and no female cells, allowing each transplant to be assessed at the individual cell level. The technique provides unique opportunities to follow the path taken by transplanted cells, both during homing into the marrow and through their maturation and differentiation into mature, functional hemopoietic cells.

The centrosome moves out of a nuclear indentation in human lymphocytes upon activation.

Using a beta-tubulin specific antibody, centrosomes were labeled in paraformaldehyde fixed human lymphocytes. Cells were kept in suspension to preserve the three-dimensional (3D) morphology as much as possible. The centrosome was generally identified as the focus of the microtubule array. Resting (G0) and phytohemagglutinin activated cells in G1 stage were taken for 3D analysis of the centrosome position, using confocal microscopy and 3D analysis software. Measurements were performed in relation to the nuclear center and the periphery of the propidium iodide stained area ("nuclear envelope"). The distribution of the distances between the centrosome and the nuclear center revealed that in most resting cells the centrosome was located at the basis of a nuclear indentation. Upon activation, however, the centrosome appeared to move out of the indentation during transition from G0 to G1 stage.

The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G0 and non-random in G1 human lymphocytes.

The nuclear topography of pericentromeric DNA of chromosome 11 was analyzed in G0 (nonstimulated) and G1 [phytohemagglutinin (PHA) stimulated] human lymphocytes by confocal microscopy. In addition to the nuclear center, the centrosome was used as a second point of reference in the three-dimensional (3D) analysis. Pericentromeric DNA of chromosome 11 and the centrosome were labeled using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence. To preserve the 3D morphology of the cells, these techniques were performed on whole cells in suspension. Three-dimensional images of the cells were analyzed with a recently developed 3D software program (Interactive Measurement of Axes and Positioning in 3 Dimensions). The distribution of the chromosome 11 centromeres appeared to be random during the G0 stage but clearly non-random during the G1 stage, when the nuclear center was used as a reference point. Further statistical analysis of the G1 cells revealed that the centromeres were randomly distributed in a shell underlying the nuclear membrane. A topographical relationship between the centrosome and the centromeres appeared to be absent during the G0 and G1 stages of the cell cycle.

Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.

Spatial analysis of intranuclear human repetitive DNA regions by in situ hybridization and digital fluorescence microscopy.

Non-isotopic (fluorescent) in situ hybridization has established itself as a useful technique for the localization of DNA sequences in both metaphase and interphase cells. The rapid development of digital fluorescence microscopy, especially confocal microscopy, has become a powerful aid for the evaluation of the hybridization results in cytogenetic and cell biological applications. In this review we will demonstrate the utility of these methodologies for the three-dimensional visualization and analysis of chromosome-specific (peri)centromeric repetitive DNA sequences within the intranuclear structure of human cells and cell lines.