Showing papers by "Diane S. Krause published in 2007"

Limitations of Green Fluorescent Protein as a Cell Lineage Marker

Abstract: The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage Here, we compare three transgenic mouse strains in which GFP is considered "ubiquitously expressed," with the GFP transgene under control of the chicken beta-actin (CBA) or human ubiquitin C (UBC) promoter We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available anti-GFP antibodies Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney UBC-GFP mice express GFP only weakly in solid organs and variably in blood Direct fluorescent detection of GFP in formalin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine In conclusion, there is considerable variability of expression within and between GFP transgenic strains None of the tested strains gave truly ubiquitous GFP expression A detailed analysis of GFP expression in one's tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin Disclosure of potential conflicts of interest is found at the end of this article

Rbm15 modulates Notch-induced transcriptional activation and affects myeloid differentiation.

Abstract: RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJkappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJkappa.

Bone marrow contributes to epithelial cancers in mice and humans as developmental mimicry.

Abstract: Bone marrow cells have the capacity to contribute to distant organs. We show that marrow also contributes to epithelial neoplasias of the small bowel, colon, and lung, but not the skin. In particular, epithelial neoplasias found in patients after hematopoietic cell transplantations demonstrate that human marrow incorporates into neoplasias by adopting the phenotype of the surrounding neoplastic environment. To more rigorously evaluate marrow contribution to epithelial cancer, we employed mouse models of intestinal and lung neoplasias, which revealed specifically that the hematopoietic stem cell and its progeny incorporate within cancer. Furthermore, this marrow involvement in epithelial cancer does not appear to occur by induction of stable fusion. Whereas previous claims have been made that marrow can serve as a direct source of epithelial neoplasia, our results indicate a more cautionary note, that marrow contributes to cancer as a means of developmental mimicry. Disclosure of Potential Conflicts of Interest is found at the end of this article.

Lung-specific nuclear reprogramming is accompanied by heterokaryon formation and Y chromosome loss following bone marrow transplantation and secondary inflammation

Abstract: Cell fusion is one mechanism by which bone marrow-derived cells (BMDCs) take on the gene expression pattern of nonhematopoietic cells. This process occurs in a number of organs with postengraftment injury but has never been found in the lung. We performed bone marrow (BM) transplant in a murine model of lung inflammation to test whether transplanted BMDCs develop lung-specific gene expression by fusing with diseased pneumocytes. Mice lacking the lung-specific protein surfactant protein C (Sp-C) were lethally irradiated, transplanted with sex mismatched wild-type marrow, and sacrificed 6 months later. Nineteen/38 recipients exhibited Sp-C mRNA (RT-PCR) and/or protein (mean 0.95±1.18 Sp-C+ cells per 1000 type II pneumocytes by confocal microscopy). In male recipients of female BM, 65% of Sp-C + cells contained the Y chromosome, indicating their origin from fusion. Only 28% of Sp-C+ cells in female recipients of male BMDCs contained the Y chromosome, suggesting that 72% of Sp-C-expressing cells lost the Y ch...

The commonly used β-actin-GFP transgenic mouse strain develops a distinct type of glomerulosclerosis

Abstract: Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We observed that the commonly used β-actin-GFP transgenic mouse has renal defects with proteinuria starting as early as 5 weeks of age. Histological analysis reveals a widespread increase in glomerular extracellular matrix, occasional mesangiolysis, and secondary tubulointerstitial injury. Electron microscopic (EM) analysis reveals dramatic thickening of the glomerular basement membrane (GBM). Several other transgenic strains with GFP on ubiquitous promoters including β-actin (with insertion in a different location) and ubiquitin C show no renal abnormalities. Western blot analysis on crude glomerular preparations from several GFP transgenic strains revealed that higher levels of GFP expression might be responsible for the observed pathogenesis. Mapping of the transgene insertion site by inverse PCR indicates that the β-actin GFP transgene does not cause insertional mutagenesis nor does it modify the transcription level of adjacent genes. Taken together, this strain of β-actin-GFP transgenic mouse may be used to study the mechanism of GBM expansion. Moreover, experiments using this strain of GFP mouse should be hereafter carefully planned because its renal pathology may interfere with data interpretation.

Circulating stem cells in extremely preterm neonates

Abstract: Aim: To measure circulating CD34+ cell levels in premature neonates and to correlate the initial CD34+ counts with measures of pulmonary function and neonatal morbidity. Methods: CD34+ cell counts were measured in the peripheral blood of preterm neonates (gestational ages 24–32 weeks) ventilated for respiratory disease at <48 h of life, and at the start of the 2nd, 3rd and 4th weeks of life. Data pertaining to neonatal demographics and short-term outcomes were collected. Pulmonary function tests were performed to coincide with CD34+ sampling. Results: Thirty preterm neonates with median gestational age of 24 weeks and birth weight of 641 g were analysed. A mean of 99.4 CD34+ cells per microliter was observed in the 1st week of life with a decline to 54.4 cells per microliter by the 4th week. An inverse correlation between initial CD34+ count and gestational age (p = 0.01) was observed. No significant correlations were observed with measures of pulmonary function or neonatal morbidities. Conclusions: Extremely premature neonates have remarkably high levels of CD34+ cells in their peripheral blood at birth. Umbilical cord blood from this population may potentially provide an abundant source of hematopoietic stem and progenitor cells for therapeutic purposes.