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Expression profiles were generated for the haemopoietic tyrosine kinase receptors (HGF-TKRs or class III TKRs) by PCR on cDNA samples (RT-PCR) using a degenerate primer set. Each profile consisted of primary and secondary, i.e. enriched for less-expressed sequences, fingerprints. This method was applied on FACS-purified haemopoietic CD34+ cells, both from bone marrow (BM) and umbilical cord blood (UCB), and on mature cells from peripheral blood. CD34+ BM cells showed expression of c-fms. flt3, whereas CD34+ UCB cells expressed c-fms and, to a lesser extent, c-kit and flt3. In mature blood cells, only c-fms was observed in monocytes and a weaker flt3 expression in monocytes and T lymphocytes, whereas no known class III TKRs were detected in B lymphocytes and polymorphonuclear cells (PMNs). In all fractions a novel band could be observed, which appeared to be RET. Expression of RET was confirmed by RT-PCR and showed the highest levels in monocytes, followed by PMNs and CD34+ cells. B lymphocytes revealed low levels of expression. RET is known to be essential in neural development. Our results suggest a possible role for this receptor in haemopoiesis.

Expression of the tyrosine kinase receptor RET has previously been detected in normal hematopoietic cells, and especially in cells of the myeloid lineage. Furthermore, RET was shown to be differentially expressed in acute myeloid leukemia (AML), a disease characterized by excessive cell growth and aberrant maturation of cells, with the highest levels of expression in leukemias with monocytic differentiation. RET is known to be expressed in cells from the excretory system and from the developing central and peripheral nervous system. Both activating and inactivating aberrations in the RET gene have been detected in disorders derived from these tissues. To investigate whether the differential expression is a primary defect in AML, the presence of RET alterations was scanned by Southern blot analysis on DNA of blasts obtained from 17 AML patients. However, no RET gene aberrations were found. Subsequently, denaturing gradient gel electrophoresis (DGGE) analysis was performed on the DNA of blasts from ten selected cases. All five variants detected turned out to represent neutral DNA polymorphisms, including a novel polymorphism in exon 14. Since we were unable to detect mutations of RET in AML, it is unlikely that it plays an important role in leukemogenesis.

SummaryOver a decade ago the idea of endothelial cell seeding was introduced in an attempt to improve the function of small caliber vascular prostheses. Although endothelial cell seeding is currently being applied clinically, several questions regarding the functional properties of the seeded endothelial cells remain. Evaluation of functional properties of endothelial cells on various types of vascular prostheses can be performed partly in vitro, but it is hampered by the fact that commonly used methods to quantify endothelial cells do not adequately apply to these cells on prosthetic materials.An accurate quantification method is described that is rapidly and easily applicable to endothelial cells attached to vascular prostheses. The method can also be used to quantify endothelial cells attached to culture dishes or microcarriers. Colorless, non-fluorescing, fluorescein-di-acetate was used, which was taken up by the attached endothelial cells, and which was then intracellularly converted to yellow fluorescein, emitting green fluorescence. Subsequently, triton-X-100 was appli-cated to release fluorescein and levels of fluorescence were measured with the automated aperture-defined microvolume (ADM) method, using an inverted fluorescence microscope to which a photometer was connected. The measured level of fluorescence is linearly related to endothelial cell numbers attached to prostheses. The accuracy and the reproducibility of cell countings are high.

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