In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology

Abstract Background Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. Methods Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. Results Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. Conclusion Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology

BACKGROUND: Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. METHODS: Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. RESULTS: Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. CONCLUSION: Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.

Autophagic programmed cell death in the suspensor and endosperm of Vicia faba : An ultrastructural study

Programmed cell death (PCD) is a widely spread phenomenon among multi-cellular organisms. Without the deletion of cells no longer needed, the organism will not be able to develop in a predicted way. It is now belived that all cells have the capacity to self-destruct and that the survival of the cells is depending on the repression of this suicidal programme. PCD has turned out to show similarities in many different species and there are strong indications that the mechanisms running the programme might, at least in some parts, be evolutionarily conserced. PCD is a generic term for different programmes of cell destruction, such as apoptosis and autophagic PCD. An important tool to determine if a cell is undergoing PCD is the transmitting electron microscope. The aims of my study were to find out if, and in what way, the suspensor and endosperm in Vicia faba (Broad bean), which are short-lived structures, undergoes PCD. The endosperm degradation preceed the suspensor cell death and they differ to some extent ultrastructurally. The cell death occurs in both tissues about 13-14 days after pollination when the embryo proper is mature enough to support itself. It was found that both tissues are committed to autophagic PCD, a cell death characteristic of conspicuous formations of autophagic vacuoles. It was shown by histochemical staining that acid phosphatases are accumulated in these vacuoles but are also present in the cytoplasm. These vacuoles are similar to autophagic vacuoles formed in rat liver cells, indicating that autophagy is a widely spread phenomenon. DNA fragmentation is the first visible sign of PCD in both tissues and it is demonstrated by a labelling technique (TUNEL). In the endosperm nuclei the heterochromatin subsequently appears in the form of a network, while in the suspensor it is more conspicuous, with heterochromatin that forms large electron dense aggregates located close to the nuclear envelope. In the suspensor, the plastids develop into chromoplasts with lycopene crystals at the same time or shortly after DNA fragmentation. This is probably due to the fact that the suspensor plastids function as hormone producing organelles and support the embryo proper with indispensable growth factors. Later the embryo will be able to produce its own growth factors and the synthesis of these, in particular gibberelines, might be suppressed in the suspensor. The precursors can then be used for synthesis of lycopene instead. Both the suspensor and endosperm are going through autophagic PCD, but the process differs in some respects. This is probably due the the different function of the two tissues, and that the signals that trigger the process presumably are different. The embryo proper is probably the source of the death signal affecting the suspensor. The endosperm, which has a different origin and function, might be controlling the death signal within its own cell. The death might in this case be related to the age of the cell.

An alternative B cell development program

Two major types of B cells, the antibody-producing cells of the immune system, are classically distinguished in the spleen: marginal zone (MZ) and follicular (FO). In addition, FO B cells are subdivided into FO I and FO II cells, based on the amount of surface IgM. MZ B cells, which surround the splenic follicles, rapidly produce IgM in response to blood-borne pathogens without T cell help, while T cell-dependent production of high affinity, isotype-switched antibodies is ascribed to FO I cells. The significance of FO II cells and the mechanism underlying B cell fate choices are unclear. We showed that FO II cells express more Sca1 than FO I cells and originate from a distinct B cell development program, marked by high expression of Sca1. MZ B cells can derive from the “canonical” Sca1lo pathways, as well as from the Sca1hi program, although the Sca1hi program shows a stronger MZ bias than the Sca1lo program, and extensive phenotypic plasticity exists between MZ and FO II, but not between MZ and FO I cells. The Sca1hi program is induced by hematopoietic stress and generates B cells with an Igλ-enriched repertoire. In aged mice, the canonical B cell development pathway is impaired, while the Sca1hi program is increased. Furthermore, we showed that a population of unknown function, defined as Lin-c-kit+Sca1+ (LSK-), contains early lymphoid precursors, with primarily B cell potential in vivo. Our data suggest that LSK- cells may represent a distinct precursor for the Sca1hi program in the bone marrow.

Vascular wall stem cells. Selection and conditioning of progenitors useful for cell therapy. A pathological case study

The arterial wall contains MSCs with mesengenic and angiogenic abilities. These multipotent precursors have been isolated from variously-sized human adult segments, belying the notion that vessel wall is a relatively quiescent tissue. Recently, our group identified in normal human arteries a vasculogenic niche and subsequently isolated and characterized resident MSCs (VW-MSCs) with angiogenic ability and multilineage potential. To prove that VW-MSCs are involved in normal and pathological vascular remodeling, we used a long-term organ culture system; this method was of critical importance to follow spontaneous 3-D vascular remodeling without any influence of blood cells. Next we tried to identify and localize in situ the VW-MSCs and to understand their role in the vascular remodeling in failed arterial homografts. Subsequently, we isolated this cell population and tested in vitro their multilineage differentiation potential through immunohistochemical, immunofluorescence, RT-PCR and ultrastructural analysis. From 25-30cm2 of each vascular wall homograft sample, we isolated a cell population with MSCs properties; these cells expressed MSC lineage molecules (CD90, CD44, CD105, CD29, CD73), stemness (Notch-1, Oct-4, Sca-1, Stro-1) and pericyte markers (NG2) whilst were negative for hematopoietic and endothelial markers (CD34, CD133, CD45, KDR, CD146, CD31 and vWF). MSCs derived from failed homografts (H-MSCs) exhibited adipogenic, osteogenic and chondrogenic potential but scarce propensity to angiogenic and leiomyogenic differentiation. The present study demonstrates that failed homografts contain MSCs with morphological, phenotypic and functional MSCs properties; H-MSCs are long-lived in culture, highly proliferating and endowed with prompt ability to differentiate into adipocytes, osteocytes and chondrocytes; compared with VW-MSCs from normal arteries, H-MSCs show a failure in angiogenic and leiomyogenic differentiation. A switch in MSCs plasticity could be the basis of pathological remodeling and contribute to aneurysmal failure of arterial homografts. The study of VW-MSCs in a pathological setting indicate that additional mechanisms are involved in vascular diseases; their knowledge will be useful for opening new therapeutic options in cardiovascular diseases.

Self assembled materials for solar cell application

In der vorliegenden Arbeit wurden Materialien und Aufbauten für Hybrid Solarzellen entwickelt und erforscht. rnDer Vergleich zweier bekannter Lochleitermaterialien für Solarzellen in einfachen Blend-Systemen brachte sowohl Einsicht zur unterschiedlichen Eignung der Materialien für optoelektronische Bauelemente als auch neue Erkenntnisse in Bereichen der Langzeitstabilität und Luftempfindlichkeit beider Materialien.rnWeiterhin wurde eine Methode entwickelt, um Hybrid Solarzelle auf möglichst unkomplizierte Weise aus kostengünstigen Materialien darzustellen. Die „Eintopf“-Synthese ermöglicht die unkomplizierte Darstellung eines funktionalen Hybridmaterials für die optoelektronische Anwendung. Mithilfe eines neu entwickelten amphiphilen Blockcopolymers, das als funktionelles Templat eingesetzt wurde, konnten mit einem TiO2-Precursor in einem Sol-Gel Ansatz verschiedene selbstorganisierte Morphologien des Hybridmaterials erhalten werden. Verschiedene Morphologien wurden auf ihre Eignung in Hybrid Solarzellen untersucht. Ob und warum die Morphologie des Hybridsystems die Effizienz der Solarzelle beeinflusst, konnte verdeutlicht werden. Mit der Weiterentwicklung der „Eintopf“-Synthese, durch den Austausch des TiO2-Precursors, konnte die Solarzelleneffizienz von 0.15 auf 0.4 % gesteigert werden. Weiterhin konnte die Übertragbarkeit des Systems durch den erfolgreichen Austausch des Halbleiters TiO¬2 mit ZnO bewiesen werden.rn

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

The role of SENP1 in B cell development and differentiation

SUMOylation is a highly dynamic and reversible posttranslational protein modification closely related to ubiquitination. SUMOylation regulates a vast array of different cellular functions, such as cell cycle, nuclear transport, DNA damage response, proliferation and transcriptional activation. Several groups have shown in in vitro studies how important SUMOylation is for early B cell development and survival as well as for later plasma cell differentiation. This thesis focuses on the deSUMOylation protease SENP1 and its in vivo effects on B cell development and differentiation. For this a conditional SENP1 knockout mouse model was crossed to the CD19-Cre mouse strain to generate a B cell specific SENP1 knockout mouse.rnIn our conditional SENP1ff CD19-Cre mouse model we observed normal numbers of all B cell subsets in the bone marrow. However in the spleen we observed an impairment of B cell survival, based on a 50% reduction of the follicular B cell compartment, whereas the marginal zone B cell compartment was unchanged. T cell numbers were comparable to control mice. rnFurther, impairments of B cell survival in SENP1ff CD19-Cre mice were analysed after in vivo blocking of IL7R signalling. The αIL7R treatment in mature mice blocked new B cell formation in the bone marrow and increased apoptosis rates could be observed in splenic SENP1 KO B cells. Additionally, a higher turnover rate of B cells was measured by in vivo BrdU incorporation.rnSince it is known that the majority of transcription factors that are important for the maintenance of the germinal centre reaction or for induction of plasma cell development are SUMOylated, the question arose, how defective deSUMOylation will manifest itself in these processes. The majority of in vitro cultured splenic B cells, stimulated to undergo class switch recombination and plasma cell differentiation underwent activation induced cell death. However, the surviving cells increasingly differentiated into IgM expressing plasma cells. Class switch recombination to IgG1 was reduced. These observations stood in line with observation made in in vivo sheep red blood cell immunization experiments, which showed increased amounts of germinal centres and germinal centre B cells, as well as increased amounts of plasma cells differentiation in combination with decreased class switch to IgG1.rnThese results lead to the conclusion that SENP1 KO B cells increasingly undergo apoptosis, however, B cells that survive SENP1 deficiency are more prone to undergo plasma cell differentiation. Further, the precursors of these plasma cells either are not as capable of undergoing class switch recombination or they do switch to IgG1 and succumb to activation induced cell death. One possible explanation for both scenarios could be a defective DNA damage response mechanisms during class switch recombination, caused by impaired deSUMOylation. rn

Differential cell cycle and proliferation marker expression in ductal pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia (PanIN)

Pancreatic cancer is an aggressive tumour following a multistep progression model through precursors called pancreatic intraepithelial neoplasia (PanIN). Identification of reliable prognostic markers would help in improving survival. The aim of this study was to investigate the role as well as the prognostic significance of different cell cycle and proliferation markers, namely p21, p27, p53 and Ki-67, in pancreatic carcinogenesis.

Blebbing confers resistance against cell lysis

The plasma membrane constitutes a barrier that maintains the essential differences between the cytosol and the extracellular environment. Plasmalemmal injury is a common event during the life of many cells that often leads to their premature, necrotic death. Blebbing - a display of plasmalemmal protrusions - is a characteristic feature of injured cells. In this study, we disclose a previously unknown role for blebbing in furnishing resistance to plasmalemmal injury. Blebs serve as precursors for injury-induced intracellular compartments that trap damaged segments of the plasma membrane. Hence, loss of cytosol and the detrimental influx of extracellular constituents are confined to blebs that are sealed off from the cell body by plugs of annexin A1 - a Ca(2+)- and membrane-binding protein. Our findings shed light on a fundamental process that contributes to the survival of injured cells. By targeting annexin A1/blebbing, new therapeutic approaches could be developed to avert the necrotic loss of cells in a variety of human pathologies.

Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles

The scope of this work was to examine in vitro responses of lung cells to secondary organic aerosol (SOA) particles, under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals, using a novel particle deposition chamber. The cell cultures included cell types that are representative for the inner surface of airways and alveoli and are the target cells for inhaled particles. The results demonstrate that an exposure to SOA at ambient-air concentrations of about 10(4) particles/cm(3) for 2 h leads to only moderate cellular responses. There is evidence for (i) cell type specific effects and for (ii) different effects of SOA originating from anthropogenic and biogenic precursors, i.e. 1,3,5-trimethylbenzene (TMB) and alpha-pinene, respectively. There was no indication for cytotoxic effects but for subtle changes in cellular functions that are essential for lung homeostasis. Decreased phagocytic activity was found in human macrophages exposed to SOA from alpha-pinene. Alveolar epithelial wound repair was affected by TMB-SOA exposure, mainly because of altered cell spreading and migration at the edge of the wound. In addition, cellular responses were found to correlate with particle number concentration, as interleukin-8 production was increased in pig explants exposed to TMB-SOA with high particle numbers.

Deregulated ephrin-B2 signaling in mammary epithelial cells alters the stem cell compartment and interferes with the epithelial differentiation pathway

Cancer most probably originates from stem/progenitor cells and exhibits a similar cell hierarchy as normal tissues. Moreover, there is growing evidence that only the stem cells are capable of metastasis formation. We have previously shown that overexpression of a dominant negative ephrin-B2 mutant interferes with mammary gland differentiation and confers a metastatic phenotype to NeuT-induced mammary tumors with an increase in cells with stem/progenitor characteristics. To investigate the role of ephrin-B2 in the control of the mammary stem cell niche, we analyzed the mammary stem and progenitor cell populations in transgenic mice overexpressing the mutant ephrin-B2. Quantification by FACS analysis revealed a significant increase of cells in the basal/alveolar cell-, the bi-potent progenitor- and the stem cell-enriched fractions. Moreover, the supposed precursors of estrogen receptor-positive cells were elevated in the stem cell-enriched fraction. In contrast, the epithelium from transgenic mice overexpressing the native ephrin-B2 gene showed an augmentation of the luminal cell- and the bi-potent progenitor-enriched fractions. Repopulation assays revealed that the epithelial cells of truncated ephrin-B2 transgenic epithelial cells have a higher regeneration capacity than those of controls and of native ephrin-B2 transgenic mice, confirming the augmentation of stem cells. Morphologically, these outgrowths exhibited impaired basal/luminal compartmentalization and epithelial polarization. These results demonstrate that deregulated ephrin-B2 expression interferes with the regulation of the stem cell niche and leads to a shift of the differentiation pathway and may thereby contribute to the acquisition of the metastatic phenotype long before carcinogenic growth becomes apparent.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches. Copyright © 2012 John Wiley & Sons, Ltd.

Hematopoietic and endothelial progenitor cell trafficking in patients with myeloproliferative diseases

BACKGROUND AND OBJECTIVES. The presence of circulating hematopoietic progenitor cells in patients with myeloproliferative diseases (MPD) has been described. However, the exact nature of such progenitor cells has not been specified until now. The aim of this work was to investigate the presence of endothelial precursor cells in the blood of patients with MPD and to assess the role of the endothelial cell lineage in the pathophysiology of this disease. DESIGN AND METHODS. Endothelial progenitor cell marker expression (CD34, prominin (CD133), kinase insert domain receptor (KDR) or vascular endothelial growth factor receptor 2 (VEGFR2), and von Willebrand factor) was assessed in the blood of 53 patients with MPD by quantitative polymerase chain reaction. Clonogenic stem cell assays were performed with progenitor cells and monocytes to assess differentiation towards the endothelial cell lineage. The patients' were divided according to whether they had essential thrombocythemia (ET, n=17), polycythemia vera (PV, n=21) or chronic idiopathic myelofibrosis (CIMF, n=15) and their data compared with data from normal controls (n=16) and patients with secondary thrombo- or erythrocytosis (n=17). RESULTS. Trafficking of CD34-positive cells was increased above the physiological level in 4/17 patients with ET, 5/21 patients with PV and 13/15 patients with CIMF. A subset of patients with CIMF co-expressed the markers CD34, prominin (CD133) and KDR, suggesting the presence of endothelial precursors among the circulating progenitor cells. Clonogenic stem cell assays confirmed differentiation towards both the hematopoietic and the endothelial cell lineage in 5/10 patients with CIMF. Furthermore, the molecular markers trisomy 8 and JAK2 V617F were found in the grown endothelial cells of patients positive for trisomy 8 or JAK2 V617F in the peripheral blood, confirming the common clonal origin of both hematopoietic and endothelial cell lineages. INTERPRETATION AND CONCLUSIONS. Endothelial precursor cells are increased in the blood of a subset of patients with CIMF, and peripheral endothelial cells bear the same molecular markers as hematopoietic cells, suggesting a primary role of pathological endothelial cells in this disease.