We have identified, cultured, characterized, and propagated adult pluripotent stem cells (PSC) from a subset of human peripheral blood monocytes. These cells, which in appearance resemble fibroblasts, expand in the presence of macrophage colony-stimulating factor and display monocytic and hematopoietic stem cell markers including CD14, CD34, and CD45. We have induced these cells to differentiate into mature macrophages by lipopolysaccharide, T lymphocytes by IL-2, epithelial cells by epidermal growth factor, endothelial cells by vascular endothelial cell growth factor, neuronal cells by nerve growth factor, and liver cells by hepatocyte growth factor. The pluripotent nature of individual PSC was further confirmed by a clonal analysis. The ability to store, expand, and differentiate these PSC from autologous peripheral blood should make them valuable candidates for transplantation therapy. P luripotent stem cells (PSC) are a valuable resource for research, drug discovery, and transplantation (1, 2). These cells or their mature progeny can be used to study differentiation processes, identify and test lineage-specific drugs, or replace tissues damaged by a disease. However, the use of PSC from human fetuses, umbilical cords, or embryonic tissues derived from in vitro fertilized eggs raises ethical and legal questions, poses a risk of transmitting infections, and͞or may be ineffective because of immune rejection. A way to circumvent these problems is by exploiting autologous stem cells, preferably from an accessible tissue. In this context, it has been reported that bone marrow contains cells that appear to have the ability to transdifferentiate into mature cells belonging to distinct cell lineages (2). A recent study indicated that bone marrow mesenchymal PSC can be expanded in vitro and after transplantation differentiate in vivo into cells belonging to distinct lineages (3). Other studies have, however, raised the possibility that such mature cells may result from fusion of stem cells with mature resident tissue cells (4,5).In the present studies, we have described the characterization and expansion in vitro of a yet unidentified subset of human peripheral blood monocytes that behave as PSC. We have shown that these cells can be induced to acquire macrophage, lymphocyte, epithelial, endothelial, neuronal, and hepatocyte phenotypes in the absence of a fusion with preexisting mature tissue cells. The ability to obtain these PSC from an easily accessible source such as peripheral blood and to store them in liquid nitrogen should make them valuable candidates for autologous transplantation. Materials and MethodsCell Culture. Monocytes were obtained from buffy coats (each from 500 ml of peripheral blood) of healthy individuals (LifeSource Blood Services, Glenview, IL) by using a selective attachment procedure as described (6, 7). Fresh mononuclear cells for this procedure and͞or after storage in liquid nitrogen in FBS (Harlan Breeders, Indianapolis) containing 10% dimethyl sulfoxide (Sigma) were obtained after Ficoll-...

SUMMARY1. Angiogenesis, the formation of new capillaries from existing vasculature, is a critical process in normal physiology as well as several physiopathologies. A desire to curb the supportive role angiogenesis plays in the development and metastasis of cancers has driven exploration into anti-angiogenic strategies as cancer therapeutics. Key to this, angiogenesis additionally displays an exquisite sensitivity to bioavailable copper. Depletion of copper has been shown to inhibit angiogenesis in a wide variety of cancer cell and xenograft systems. Several clinical trials using copper chelation as either an adjuvant or primary therapy have been conducted. Yet, the biological basis for the sensitivity of angiogenesis remains unclear. Numerous molecules important to angiogenesis regulation have been shown to be either directly or indirectly influenced by copper, yet a clear probative answer to the connection remains elusive. 2.Measurements of copper in biological systems have historically relied on techniques that, although demonstrably powerful, provide little or no information as to the spatial distribution of metals in a cellular context. Therefore, several new approaches have been developed to image copper in a biological context. One such approach relies on synchrotron-derived X-rays from third-generation synchrotrons and the technique of high resolution X-ray fluorescence microprobe (XFM) analysis.3. Recent applications of XFM approaches to the role of copper in regulating angiogenesis have provided unique insight into the connection between copper and cellular behaviour. Using XFM, copper has been shown to be highly spatially regulated, as it is translocated from perinuclear areas of the cell towards the tips of extending filopodia and across the cell membrane into the extracellular space during angiogenic processes. Such findings may explain the heightened sensitivity of this cellular process to this transition metal and set a new paradigm for the kinds of regulatory roles that the spatial dynamics of cellular transition metals may play.

Although copper has been reported to influence numerous proteins known to be important for angiogenesis, the enhanced sensitivity of this developmental process to copper bioavailability has remained an enigma, because copper metalloproteins are prevalent and essential throughout all cells. Recent developments in x-ray optics at third-generation synchrotron sources have provided a resource for highly sensitive visualization and quantitation of metalloproteins in biological samples. Here, we report the application of x-ray fluorescence microscopy (XFM) to in vitro models of angiogenesis and neurogenesis, revealing a surprisingly dramatic spatial relocalization specific to capillary formation of 80 -90% of endogenous cellular copper stores from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane. Although copper chelation had no effect on process formation, an almost complete ablation of network formation was observed. XFM of highly vascularized ductal carcinomas showed copper clustering in putative neoangiogenic areas. This use of XFM for the study of a dynamic developmental process not only sheds light on the copper requirement for endothelial tube formation but highlights the value of synchrotron-based facilities in biological research.copper chelation ͉ human microvascular endothelial cells ͉ infiltrating ductal breast carcinoma E ndogenous metals, such as Cu, Fe, and Zn, are subject to complex regulation in cellular systems. They are required as cofactors or regulators of numerous proteins (1) and yet, if present in overabundance, are toxic and expose the cellular environment to adventitious redox activity (2). This delicate balance is thought to be achieved by sequestration of these metals within their target proteins, metallochaperone systems, or distinct subcellular compartments (3). Although many proteins that handle transition metals within cells have been identified (4), our knowledge as to how metal content is dynamically regulated in eukaryotic cells is still limited. To what extent does regulation of the metal ion content of individual metalloproteins, mediated by protein-protein interactions, serve as an additional level of regulation of cellular metalloprotein activity? Could such regulation result in polarization of transition metal distribution throughout a cell during a biological process? To begin to explore such questions, we examined a cellular system whose biology is acutely sensitive to modulation by metals.

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-␤ (PKC-␤) and several stable PKC-␤ transfectants, we found that PRKX gene expression is under control of PKC-␤; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of ceHlular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time-and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.Cellular systems have mechanisms to regulate the expression of genes that mediate the synthesis of necessary metabolites. These mechanisms must respond to internal and external factors in such a way that an appropriate supply of these metabolites is provided for the cell or organism. In prokaryotic systems, some genes are regulated at the transcriptional level by metabolic end products. In Bacillus subtilis, the pur operon encodes enzymes involved in purine biosynthesis, 11 of which catalyze the formation of IMP from 5-phosphoribosyl-1-pyrophosphate (13). This operon is regulated by the purine end products, adenine and guanine nucleotides. Although end-product regulation of genes is common in prokaryotic systems, no parallel has been reported in the regulation of eukaryotic nucleotide biosyntheSiS. The steady-state mRNA level of human IMP dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme in de novo guanine nucleotide biosynthesis, is elevated in human HL-60 and CEM leukemia and HO melanoma cells after they are treated with the competitive IMPDH inhibitor mycophenolic acid (MPA) (11,20,21). This induction is preceded by a decrease in the level of cellular guanine nucleotides as a consequence of MPA-mediated inhibition of IMPDH enzyme activity. These observations suggest that the level of purine end products may influence the expression of the IMPDH gene, perhaps in a manner similar to that observed for the pur operon genes in B. subtilis. To examine the control of IMPDH gene expression, we treated human and Chinese hamster cell lines with nucleosides, nucleotides, nucleotide analogs, or MPA and analyzed the effect on the expression of the IMPDH gene. We provide evidence that the expression of this gene is indeed regulated by guanine ribonucleo...

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