Stem cells reside in a specialized niche that regulates their abundance and fate. Components of the niche have generally been defined in terms of cells and signaling pathways. We define a role for a matrix glycoprotein, osteopontin (OPN), as a constraining factor on hematopoietic stem cells within the bone marrow microenvironment. Osteoblasts that participate in the niche produce varying amounts of OPN in response to stimulation. Using studies that combine OPN-deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell number and function in a stem cell–nonautonomous manner. The OPN-null microenvironment was sufficient to increase the number of stem cells associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive hematopoietic cell apoptosis. The activation of the stem cell microenvironment with parathyroid hormone induced a superphysiologic increase in stem cells in the absence of OPN. Therefore, OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation.
Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amounts of information support the fact that it is also associated with a high percentage of false-positive results. In our study, we not only managed to demonstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely novel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the CK19 mRNA to exclude DNA contamination. Tumor dilution experiments were performed in order to analyze the specificity and sensitivity of the method. Control experiments using the blood of healthy donors were performed. Tumor cell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detection limit was 1 tumor cell in 105 mononuclear cells. RT-PCR of mononuclear cells from healthy blood donors gave false-positive results in 29% of the cases. We conclude that a significant decrease in the sensitivity of CK19 RT-PCR occurs if it is performed in blood cells and that the illegitimate CK19 gene expression in normal cells can lead to false-positive results. These limitations have to be taken into account if RT-PCR is to be used for the detection of tumor cells either in blood or in bone marrow in clinical practice.
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