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Cancer stem cells (CSCs) are subpopulations of cancer cells with high self-renewal potential that are involved in tumor progression and recurrence. It has been postulated that CSCs and non-stem cancer cells are inter-convertible. However, precise mechanisms for the plasticity and stability of cancer stemness remain elusive. Here, we demonstrate that CD44-positive colorectal CSC fractions contain two types of cancer cells: "CD44-stable" cells, in which CD44 expression is stably sustained, and "CD44-trasnsient" cells, which are rapidly converted to CD44-negative cells. CD44-stable cells expressed higher levels of c-KIT tyrosine kinase than CD44-transient cells. c-KIT knockdown by siRNAs converted the CD44-positive cells to CD44-negative cells, which expressed lower levels of stem cell markers such as ASCL2 and EPCAM. In the CD44-positive cells, c-KIT phosphorylation level was very low whereas stem cell factor, a c-KIT ligand, elevated c-KIT phosphorylation without affecting stem cell marker expression. CRISPR-Cas9-mediated knockout of the c-KIT gene in CD44 stable cells attenuated the CSC properties including expression of CD44 and other stem cell markers, clonogenicity and in vivo tumorigenic potential in a mouse xenograft model. These observations suggest that the colorectal CSC fractions contain cancer cells with differential plasticity, which is determined by c-KIT., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Natural killer (NK) cells are a population of lymphoid cells that play a significant role in mediating innate immune responses. Studies in mice suggest a pathological role for NK cells in models of kidney disease. In this study, we characterized the NK cell subsets present in native kidneys of patients with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. Significantly higher numbers of total NK cells (CD3 - CD56 + ) were detected in renal biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue using multi-color flow cytometry. At a subset level, both the CD56 dim NK cell subset and particularly the CD56 bright NK cell subset were elevated in fibrotic kidney tissue. However, only CD56 bright NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56 bright NK cells was significantly increased in fibrotic biopsy specimens compared with non-fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric phenotyping revealed selective co-expression of activating receptor CD335 (NKp46) and differentiation marker CD117 (c-kit) on CD56 bright NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56 bright NK cells (NKp46 + CD117 + ) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ-producing CD56 bright NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

In this issue of Blood, Dahlin et al report on a minor circulating human mast cell (MC) progenitor cell population (lineage-negative [Lin−]/CD34hi/CD117int/hi/high-affinity immunoglobulin E receptor-positive [FcεRI+]), with an immature MC-like appearance, which is present in the peripheral blood (PB) of healthy individuals and of asthma subjects well controlled by treatment or with reduced lung function.

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function., (© 2016 by The American Society of Hematology.)

Mast cell tumours (MCTs) are a common skin tumour in cats, but there is currently no histological grading system or reliable prognostic marker for this species (unlike the situation for dogs). This study utilized a set of 71 feline cutaneous MCTs with known clinical outcomes to assess the potential of various prognostic markers, including the cellular proliferation marker minichromosome maintenance protein (MCM)-7, mitotic index and various KIT labelling characteristics, including KIT positivity, KIT labelling pattern and KIT immunoreactivity score (IS). Of the factors studied, the mitotic index and the KIT labelling pattern were the only features associated significantly with survival times, while the proliferation marker MCM7 and the KIT IS were not. The study also highlights the variability of KIT labelling characteristics between tumours, which may prevent use of this marker as a diagnostic and prognostic tool., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: The pathogenesis of abdominal aortic aneurysm (AAA) formation includes inflammation, vascular smooth muscle cell apoptosis, extracellular matrix degradation, and oxidative stress. That multipotent stem cells have an important role in cardiovascular health and disease has been well established, but the role of stem cells in aortic structural deterioration is poorly defined. We sought to describe the presence of stem cells in human AAA tissue and also investigated the differentiation of stem cells within the aneurysmal aorta., Methods: Infrarenal aortic wall specimens were collected from patients (n = 7) undergoing open AAA surgical repair. Nonaneurysmal infrarenal aortic control samples (n = 4) were collected at autopsies. Using immunohistochemistry, we compared the abundance of Stro1-positive ((+)), c-kit(+), and CD34(+) cells in aortic tissue. Using double-immunofluorescence staining, we evaluated stem cell differentiation into smooth muscle cells (SM22), fibroblasts (FSP1), and macrophages (CD68). We then investigated the colocalization of CD68(+) cells with the cellular marker of proliferation Ki67., Results: The media and adventitia of infrarenal AAA samples both demonstrated a significantly greater number of c-kit(+) and CD34(+) cells compared with matched control nonaneurysmal aortic tissues; however, the abundance of Stro1(+) cells was not significantly different between the groups. Using double-immunofluorescence staining, we identified that AAA stem cells express the macrophage marker CD68 but not the smooth muscle cell marker SM22 or the fibroblast marker FSP1. CD68(+) cells within the aortic wall colocalized with the cellular marker of proliferation Ki67., Conclusions: Stem cells are significantly elevated in infrarenal AAA tissue compared with matched control aortic tissue. Our data also demonstrate that AAA stem cells express macrophage surface antigens but not smooth muscle cell or fibroblast markers. Furthermore, CD68(+) cells within the aortic wall colocalized with the cellular marker of proliferation Ki67. These finding suggest an inflammatory/immune role of stem cells during AAA pathogenesis and raise the possibility of localized replenishment therapy within the aneurysm wall., (Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Chagasic cardiomyopathy, resulting from infection with the parasite Trypanosoma cruzi, was discovered more than a century ago and remains an incurable disease. Due to the unique properties of mesenchymal stem cells (MSC) we hypothesized that these cells could have therapeutic potential for chagasic cardiomyopathy. Recently, our group pioneered use of nanoparticle-labeled MSC to correlate migration with its effect in an acute Chagas disease model. We expanded our investigation into a chronic model and performed more comprehensive assays. Infected mice were treated with nanoparticle-labeled MSC and their migration was correlated with alterations in heart morphology, metalloproteinase activity, and expression of several proteins. The vast majority of labeled MSC migrated to liver, lungs and spleen whereas a small number of cells migrated to chagasic hearts. Magnetic resonance imaging demonstrated that MSC therapy reduced heart dilatation. Additionally metalloproteinase activity was higher in heart and other organs of infected mice. Protein expression analyses revealed that connexin 43, laminin γ1, IL-10 and INF-γ were affected by the disease and recovered after cell therapy. Interestingly, MSC therapy led to upregulation of SDF-1 and c-kit in the hearts. The beneficial effect of MSC therapy in Chagas disease is likely due to an indirect action of the cells of the heart, rather than the incorporation of large numbers of stem cells into working myocardium., (Copyright © 2014. Published by Elsevier Masson SAS.)

Introduction: The 2004 version of the World Health Organization classification subdivides thymic epithelial tumors into A, AB, B1, B2, and B3 (and rare other) thymomas and thymic carcinomas (TC). Due to a morphological continuum between some thymoma subtypes and some morphological overlap between thymomas and TC, a variable proportion of cases may pose problems in classification, contributing to the poor interobserver reproducibility in some studies., Methods: To overcome this problem, hematoxylin-eosin-stained and immunohistochemically processed sections of prototypic, "borderland," and "combined" thymomas and TC (n = 72) were studied by 18 pathologists at an international consensus slide workshop supported by the International Thymic Malignancy Interest Group., Results: Consensus was achieved on refined criteria for decision making at the A/AB borderland, the distinction between B1, B2, and B3 thymomas and the separation of B3 thymomas from TCs. "Atypical type A thymoma" is tentatively proposed as a new type A thymoma variant. New reporting strategies for tumors with more than one histological pattern are proposed., Conclusion: These guidelines can set the stage for reproducibility studies and the design of a clinically meaningful grading system for thymic epithelial tumors.

C-kit expressing cardiac stem cells have been described as multipotent. We have previously identified human cardiac C-kit+CD45- cells, but only found evidence of endothelial commitment. A small cardiac committed subpopulation within the C-kit+CD45- population might however be present. To investigate this at single-cell level, right and left atrial biopsies were dissociated and analyzed by FACS. Only right atrial biopsies contained a clearly distinguishable C-kit+CD45- population, which was single-cell sorted for qPCR. A minor portion of the sorted cells (1.1%) expressed early cardiac gene NKX2.5 while most of the cells (81%) expressed late endothelial gene VWF. VWF- cells were analyzed for a wider panel of genes. One group of these cells expressed endothelial genes (FLK-1, CD31) while another group expressed late cardiac genes (TNNT2, ACTC1). In conclusion, human C-kit+CD45- cells were predominantly localized to the right atrium. While most of these cells expressed endothelial genes, a minor portion expressed cardiac genes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: CXC ligand 12/stromal-derived factor-1 (CXCL12/SDF-1) is a pleiotropic chemokine that regulates the influx of a wide range of leukocytes. The aim of this study was to characterize CXCL12/SDF-1 in apical lesions (ALs) of endodontic origin, with special emphasis in associated immune cell populations., Methods: In this case-control study, 29 individuals with chronic apical periodontitis and 21 healthy volunteers were enrolled. ALs and healthy periodontal ligament samples were obtained for tissue homogenization, immune Western blotting, and enzyme-linked immunosorbent assay to determine CXCL12/SDF-1 forms and levels. Anatomopathologic diagnosis, immunostaining for CXCL12/SDF-1, CD117-CXCL12/SDF-1, and toluidine blue were also performed to identify tissue and cell localization. Finally, a set of tissue samples were digested and analyzed by flow cytometry to identify CXCL12/SDF-1 in different immune cell populations. Data were analyzed with Stata v11 and WinDi 2.9 software, and significance was considered if P < .05., Results: CXCL12/SDF-1 was predominantly identified as monomers; levels of CXCL12/SDF-1 were significantly higher in ALs compared with controls, and it was primarily localized to inflammatory infiltrates. Expression of CXCL12/SDF-1 was colocalized to mast cells in tissue sections. Furthermore, CD117(+) mast cells were the second most frequent infiltrating cells and the main CXCL12/SDF-1 expressing cells, followed by CD4(+) lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells., Conclusions: ALs of endodontic origin demonstrated higher levels of CXCL12/SDF-1 compared with controls. CXCL12/SDF-1 was identified in immune cell populations, whereas mast cells represented the major CXCL12/SDF-1 expressing cells, suggesting that this chemokine might play a central role in apical tissue destruction, most probably inducing persistent recruitment of immune cells, particularly of mast cells., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

White adipose tissue (WAT) is the focus of new interest because of the presence of an abundant and complex immune cell population that is involved in key pathologies such as metabolic syndrome. Based on in vivo reconstitution assays, it is thought that these immune cells are derived from the bone marrow (BM). However, previous studies have shown that WAT exhibits specific hematopoietic activity exerted by an unknown subpopulation of cells. In the present study, we prospectively isolated a peculiar hematopoietic stem/progenitor cell population from murine WAT. The cells are phenotypically similar to BM hematopoietic stem cells and are able to differentiate into both myeloid and lymphoid lineages in vitro. In competitive repopulation assays in vivo, they reconstituted the innate immune compartment in WAT preferentially and more efficiently than BM cells, but did not reconstitute hematopoietic organs. They were also able to give rise to multilineage engraftment in both secondary recipients and in utero transplantation. Therefore, we propose that WAT hematopoietic cells constitute a population of immature cells that are able to renew innate immune cell populations. Considering the amount of WAT in adults, our results suggest that WAT hematopoietic activity controls WAT inflammatory processes and also supports innate immune responses in other organs.

Introduction: We have previously differentiated hepatocyte like cells from deciduous tooth pulp stem and extracted third molar pulp stem cells with a protocol that used fetal bovine serum, but it showed high contaminations of nondifferentiated cells. Both the lower purity of hepatically differentiated cells and usage of serum are obstacles for application of cell therapy or regenerative medicine. Objective of this study was to investigate the capacity for and purity of hepatocyte-like differentiation of CD117-positive dental pulp stem cells without serum., Methods: Mesenchymal cells from human deciduous and extracted third molar pulp were isolated and expanded in vitro. We separated CD117-positive cells by using a magnetic-activated cell sorter. The cells were characterized immunofluorescently by using known stem cell markers. For hepatic differentiation, the media were supplemented with hepatic growth factor, insulin-transferrin-selenium-x, dexamethasone, and oncostatin M. Expression of hepatic markers alpha fetoprotein, albumin, hepatic nuclear factor-4 alpha, insulin-like growth factor-1, and carbamoyl phosphate synthetase was examined immunofluorescently after differentiation. The amount of differentiated cells was assessed by using flow cytometry. Glycogen storage and urea concentration in the medium were defined., Results: Both cell cultures demonstrated a number of cells positive for all tested hepatic markers after differentiation, ie, albumin-positive cells were almost 90% of differentiated deciduous pulp cells. The concentration of urea in the media increased significantly after differentiation. Significant amount of cytoplasmic glycogen storage was found in the cells., Conclusions: Without serum both cell types differentiated into high-purity hepatocyte-like cells. These cells offer a source for hepatocyte lineage differentiation for transplantation in the future., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Dental pulp stem cells (DPSCs) have received much attention as a promising population of stem cells in regenerative endodontics. Securing a good blood supply during regeneration is a challenging task because of the constricted apical canal opening, which allows only a limited blood supply. The aim of this study was to investigate any potential synergistic effects of dental pulp stem cells and endothelial cells (ECs) on osteo-/odontogenic and angiogenic differentiation in vitro., Methods: Different ratios of DPSCs and ECs were cultured in direct contact using optimized medium for coculture. The 70% confluent cocultures were incubated in the osteo-/odontogenic differentiation medium for up to 3 weeks. Alkaline phosphatase (ALP) activity, the expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes, and alizarin red staining for mineralization at different time points were analyzed. The tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34, and Flk-1 were used as assays to analyze angiogenesis., Results: The quantification of ALP in DPSC:EC cocultures revealed a greater ALP activity compared with DPSC-alone cultures. At all the time points, 1:1 cultures showed a significantly greater ALP activity than that of DPSC-alone cultures. Alizarin red staining and quantification revealed a much greater amount of calcification in the 1:1 and 1:5 cocultures compared with other cultures (P < .01). The expression levels of ALP, BSP, and DSPP genes further confirmed the greater osteo-/odontogenic differentiation in cocultures compared with those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized preexisting vessel-like structures formed by ECs and increased the longevity of them., Conclusions: Direct coculture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes., (Copyright © 2012 American Association of Endodontists. All rights reserved.)

Aims: Coronary thrombotic occlusion in ST-segment elevation myocardial infarction (STEMI) patients is often preceded by episodes of progressive growth of the thrombus mass. Similar to wound healing, the organization of thrombus could depend on ingrowth of microvessels in order to stabilize its structure. We investigated the patterns of neovascularization in different stages of coronary thrombus evolution., Material and Methods: Thrombectomy materials obtained from STEMI patients were histologically classified according to thrombus age in three groups: fresh (< 1 day), lytic (1-5 days) or organized (> 5 days) thrombi. Forty thrombi of each group were randomly collected. Neovascularization in the thrombi was evaluated histomorphologically and with immunodouble stains to visualize various differentiation antigens of endothelial cells (ECs) and primitive cells., Results: Morphologically, ECs in the coronary thrombi manifested as: single cells, cell clusters or microvessels. CD31+/CD34+ ECs were present in 98% of all the thrombi. In addition, endothelial clusters were found in 63% of the fresh thrombi (< 1 day). CD105+, Ki67+, or C-kit+ ECs (active, proliferating cells) were observed in all the stages, but significantly more in organized thrombi (> 5 days) compared with fresh and lytic ones (< 5 days), and mainly as cell clusters (P ≤ 0.05 for all). CD133+ primitive cells were found only sporadically in 11% of all the samples., Conclusion: EC proliferation is initiated very early, and gradually progresses during the organization process of thrombus after coronary plaque disruption, with only a limited contribution of primitive cells in this process., (© 2012 International Society on Thrombosis and Haemostasis.)

Mixed chimerism has been suggested to induce tolerance to transplanted alloantigens. As the precise influence of mixed chimerism induction on the host organism has still not been fully elucidated, the aim of the present study was to explore this phenomenon in relation to the stem cell compartment. The experiment was performed on B6.SJL-Ptprc(a)Pep3(b) mice. Mixed chimerism induction protocols involved 3 Gy TBI (Day -1 of the experiment), injection of 20-30 × 10(6) Balb C bone marrow cells (Day 0), and administration of blocking antibodies against CD40L (Day 0 and Day 4), anti-CD8 (Day -2) with/without anti-NK1.1 (Day -3). Selected groups of mice were also treated with cyclophosphamid (175 mg/kg) on Day 2. The presence of mixed chimerism was assessed in peripheral blood, bone marrow, and spleen, as well as in various subpopulations of leukocytes (CD4(+), CD8(+), CD45/B220(+), Gr-1(+), lin(-)/Sca-1(+)/c-kit(-), lin(-)/Sca-1(+)/c-kit(+), lin(-)/Sca-1(-)/c-kit(+)). Furthermore, the percentage of stem/progenitor cells (lin(-)/Sca-1(+)/c-kit(-), lin(-)/Sca-1(+)/c-kit(+), lin(-)/Sca-1(-)/c-kit(+), VSEL, HSC) was analysed for the first time in bone marrow and peripheral blood of chimeric mice. The range of mixed chimerism differed significantly among various cell populations: it was lowest in CD8-positive cells and lin(-)/Sca-1(+)/c-kit(-) cells, and highest in granulocytes. The induction of mixed chimerism revealed a significant impact on the stem/progenitor cell frequency in recipient mice, providing potential therapeutic insights into the long-term immunologic tolerance observed in chimeric mice. Collectively, these findings contribute to further optimization of mixed chimerism induction protocols and might help in the introduction of this phenomenon into clinical practice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The size of the breast stem-cell pool could underlie the intrauterine roots of breast cancer. We studied whether breast stem cells exist in umbilical cord blood and if they correlate with hematopoietic stem-cell measurements that have been positively associated with perinatal risk factors for breast cancer., Subjects and Methods: We isolated mononuclear cells from umbilical cord blood of 170 singleton full-term pregnancies and determined, by reverse transcription polymerase chain reaction, the presence of genes of putative breast epithelial stem-cell/progenitor markers [including epithelial cell adhesion molecule (EpCAM), CD49f (α6-integrin), CD117 (c-kit receptor), CD24, and CD29 (β1-integrin)]. By immunocytochemistry, we colocalized protein expressions of EpCAM+CD49f+, CD49f+CD24+, and CD24+CD29+. We correlated concentrations of putative breast stem-cell/progenitor subpopulations, quantified by flow cytometry, with concentrations of hematopoietic stem cells., Results: Mammary stem-cell phenotypes were identified in umbilical cord blood. The measured EpCAM+ subpopulation was positively correlated with concentrations of CD34+ and CD34+CD38- hematopoietic stem cells (both P=0.006). Additionally, EpCAM+CD49f+ and CD49f+CD24+ subpopulations were positively correlated to the CD34+ cells (P=0.03 and 0.008, respectively)., Conclusion: The positive association between measurable breast and hematopoietic stem cells in human umbilical cord blood suggests plausible mechanisms for a prenatal influence on breast cancer risk.

Gastrointestinal stromal tumors (GISTs), mesenchymal neoplasms originating from the cells of Cajal, usually appear in the gastrointestinal tract and abdomen. They often mimic other lesions, including smooth muscle cell tumors and neurogenic tumors. This study presents a case in which a GIST appeared over a 2-month period and was treated by excision and curettage, with no sign of recurrence during the next 42 months. The study also aims to characterize the GIST. Histopathologic analysis and KIT gene amplification and sequencing were performed. On mutation analysis of the GIST material, the novel 69338Tdel mutation was found in exon 11, and the diagnosis of intraoral stromal tumor was made. GISTs in the intraoral region display pathologic properties similar to others developed throughout the gastrointestinal system. Diagnosis is the first step of treatment for a patient. The discovery of oncogenic KIT mutations in GISTs has led to the development of targeted molecular therapy using tyrosine kinase inhibitors. This study investigates the histopathologic and molecular diagnostics of GISTs, and, to the authors' knowledge, it represents the first genetic study of a GIST developing in the intraoral region., (Copyright © 2010 Mosby, Inc. All rights reserved.)

We report a case of a 50-year old post-menopausal woman who was admitted because of a lump in the upper external quadrant of her left breast. The definitive diagnosis of periductal stromal tumor was retained after histopathological examination. Periductal stromal tumor is a rare tumor with distinct morphological features. The clinical evolution and the prognosis are relatively similar to the phyllodes tumor., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Background: Imatinib myselate is a molecularly targeted drug that inhibits Abl tyrosine kinase, as well as type III tyrosine kinase receptors such as platelet-derived growth factor receptor (PDGFR), KIT, colony-stimulating factor 1 receptor (CSF-1R), and discoidin domain receptor (DDR). Ph1 chromosome-positive chronic myeloid leukemias (CMLs), KIT-positive gastrointestinal stromal tumors (GISTs), and PDGFR-positive dermatofibrosarcoma protuberans (DFSP) have been reported to be responsive to imatinib treatment. We conducted a multicenter Phase II trial of imatinib in patients with relapsed or refractory KIT-positive (excluding GISTs) or PDGFR-positive sarcomas., Methods: Patient ages ranged from 12 and 75 years. Eligibility criteria included (1) metastatic sarcomas with a definitive diagnosis based on histopathology or that were completely unresectable and locally advanced; (2) relapsed or refractory cases that had completed standard treatment; and (3) a tumor confirmed by immunohistochemical staining to be KIT- or PDGFR-positive. A 600-mg dose of imatinib was administered to patients once a day, with each patient receiving six courses of the drug and each course lasting 4 weeks. In cases categorized as stable or progressive, the imatinib dose was increased to 800 mg/day administered twice daily., Results: A total of 25 patients who met the eligibility criteria were enrolled in the trial; 22 were evaluated for response. The response rate with a 600 mg/day dose of imatinib was 4.5% (0 complete response, 1 partial response). There were no other objective responses after increasing imatinib to 800 mg/day (0/10). We estimated 50% progression-free survival to be 61.0 days for an imatinib dose of 600 mg/day based on the Kaplan-Meier method. Side effects of imatinib were generally similar to those observed in previous clinical trials., Conclusions: Our results did not indicate effectiveness of imatinib monotherapy at a dose of 600 or 800 mg/day in patients with relapsed or refractory KIT-positive (excluding GISTs) or PDGFR-positive sarcomas. Our findings suggest the need to evaluate the synergistic effect of combination therapy with other anticancer drugs.

Background: Advanced or recurrent nonresectable thymic epithelial tumors show only a modest response to standard chemotherapy. A recent study using octreotide and prednisone in thymic tumors, Eastern Cooperative Oncology Group study E1C97, was conducted to verify the activity of octreotide for thymic tumors. The aim of this study was to determine whether epidermal growth factor receptor (EGFR) immunoreactivity correlated with outcomes and to identify new biologic markers for potential targeted therapy. Three markers, EGFR, C-kit, and Her2/neu, were selected for evaluation in patients with advanced thymic epithelial tumors treated on E1C97., Methods: Of the 42 patients entered onto E1C97, 34 patients (World Health Organization [WHO] categories: type A = 1, type AB = 1, type B1 = 10, type B2 = 11 type B3 = 8, and type C = 3) had sufficient tissue available for immunohistologic study. Each tumor was assessed to have 0, 1+, 2+, or 3+ immunoreactivity in the cytoplasm or membranes of the neoplastic cells for Her2/neu and EGFR and for the presence or absence of C-kit immunoreactivity., Results: EGFR immunoreactivity of 2+ or 3+ was associated with more aggressive thymic tumors (WHO types B2 and B3). However, strong EGFR immunoreactivity was not consistently seen with thymic carcinoma. The presence of EGFR within cells was associated with a significantly improved progression-free survival (PFS) and a trend for overall survival (OS). Twelve patients demonstrated C-kit immunoreactivity; the lack of C-kit immunoreactivity was significantly associated with superior PFS but not OS. Her2/neu immunoreactivity was uniformly negative for all tumors evaluated. There was no association between response and biomarker status., Conclusions: High EGFR immunoreactivity is seen in more aggressive thymic neoplasms as classified according to the 2004 WHO, but regardless of classification, the presence of EGFR in tumor cells (1+, 2+, and 3+) is associated with improved performance free survival (PFS) and a trend for better OS. In contrast, the absence of C-kit immunoreactivity was associated with improved PFS. These data suggest that EGFR and C-kit may be prognostic, and further studies of these markers in subcategories of thymic malignancies is warranted.

Objectives: In view of evidence that mature cells play a role in modulating the stem cell niche and thereby stem cell potential and proliferation, we hypothesized that a mature bone marrow (BM) mononuclear cell (MNC) infusion subfraction may have particular potency in promoting hematopoietic or resident stem cell-induced cardiac repair post-infarction., Background: Treatment of acute myocardial infarction (MI) with BM MNC infusion has shown promise for improving patient outcomes. However, clinical data are conflicting, and demonstrate modest improvements. BM MNCs consist of different subpopulations including stem cells, progenitors, and differentiated leukocytes., Methods: Stem cells (c-kit+) and subsets of mature cells including myeloid lineage, B and T-cells were isolated from bone marrow harvested from isogeneic donor rats. Recipient rats had baseline echocardiography then coronary artery ligation; 1 x 10(6) cells (enriched subpopulations or combinations of subpopulations of BM MNC) or saline was injected into ischemic and ischemic border zones. Cell subpopulations were either injected fresh or after overnight culture. After 2 weeks, animals underwent follow-up echocardiography. Cardiac tissue was assayed for cardiomyocyte proliferation and apoptosis., Results: Fractional ventricular diameter shortening was significantly improved compared with saline (38 +/- 3.2%) when B cells alone were injected fresh (44 +/- 3.0%, p = 0.035), or after overnight culture (51 +/- 2.9%, p < 0.001), or after culture with c-kit+ cells (44 +/- 2.4%, p = 0.062). B cells reduced apoptosis at 48 h after injection compared with control cells (5.7 +/- 1.2% vs. 12.6 +/- 2.0%, p = 0.005)., Conclusions: Intramyocardial injection of B cells into early post-ischemic myocardium preserved cardiac function by cardiomyocyte salvage. Other BM MNC subtypes were either ineffective or suppressed cardioprotection conferred by an enriched B cell population.

Background: Imatinib mesylate is a potent inhibitor of the Bcr-Abl, c-Kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases. On the basis of variable expression of c-Kit and PDGFR in breast cancer and of in vitro data supporting synergy between imatinib and capecitabine, the Southwest Oncology Group conducted a phase II trial of the combination in metastatic breast cancer., Patients and Methods: Eligible patients had progressive, measurable metastatic breast cancer and received

There have been some proposals that stem cells exist in the ovarian surface epithelium (OSE) of the adult human ovary; however, no direct evidence of such cells has been given until now. The aim of this study was to isolate the putative ovarian stem cells (OSCs) from the OSE layer in women with no naturally present oocytes and follicles--20 postmenopausal women and five women with premature ovarian failure. Small round cells with a bubble-like structure and diameters from 2 to 4 microm were isolated from the material obtained by OSE scraping. They expressed early embryonic developmental markers such as stage-specific embryonic antigen-4 and Oct-4, Nanog, Sox-2, and c-kit transcription markers, and they displayed prominent c-kit immunohistochemical staining. These cells were separated by density gradient centrifugation and grown in vitro, where they proliferated. Some of them grew intensively and reached a diameter of approximately 20 microm after 5-7 days. In the OSE cell culture, oocyte-like cells developed, which reached a diameter of up to 95 microm and expressed Oct-4A, Oct-4B, c-kit, VASA, and ZP2 transcription markers, corresponding to early oocytes. They did not express SCP3 meiotic marker. In conclusion, the discovered cells are proposed to represent the adult OSCs with the expression of embryonic stem cell markers. The expression of germ lineage marker c-kit points toward their primordial germ cell ancestry. A new term "embryonic-like stem cells of the adult" is proposed for embryonic-like stem cells that might persist in various tissues and organs of adults. These findings could be used for further studies aimed at the autologous treatment of ovarian infertility and degenerative diseases.

We describe a rare case of leiomyosarcoma originating from the superior mesenteric vein with concomitant liver metastases in a 62-year-old woman. She underwent a tumor resection and venous reconstruction, right hemicolectomy, and right hepatic lobectomy. Since the tumor was weakly positive for c-kit, she was treated with imatinib mesylate for the recurrent liver tumors. She has survived for about 3 years after undergoing the surgical procedures.

c-kit (CD117) plays an important role in the early stages of haematopoiesis. Previous studies of porcine haematopoietic stem cells have relied for their identification on the use of the c-kit ligand stem cell factor. Here, we describe a new mAb, 2B8/BM, that recognizes a 155-kDa protein expressed on a small subset (2-8%) of bone marrow haematopoietic cells. 2B8/BM(+) cells have a blast appearance, and are mostly negative for lineage-specific markers or express low levels of CD172a or SLA-II. In in vitro colony-forming unit assays these cells were able to give rise to erythroid and myeloid colonies. Altogether these data suggested that the 2B8/BM antigen might be the porcine orthologue of the human c-kit. This specificity was confirmed by the binding of mAb 2B8/BM to CHO cells transfected with a plasmid encoding the porcine c-kit ectodomain. This antibody can facilitate the isolation and enrichment of porcine stem cells to be used in procedures aimed to induce xenograft tolerance or to test their potential to repair damaged tissues and organs.

Gastrointestinal stromal tumors (GISTs) frequently harbor mutations in the KIT and PDGFRA genes, the presence and type of which correlate with the response to the kinase inhibitor imatinib mesylate. Because most GIST mutations are deletions/insertions, we used a microfluidic apparatus to detect these size variations in polymerase chain reaction-amplified DNA. This approach, termed microfluidic deletion/insertion analysis (MIDIA), identified mutations in 30 of 50 DNA samples from paraffin-embedded CD117-positive GISTs (60%), comprising 25 deletions and five insertions. Sequencing of 14 MIDIA-positive samples confirmed the deletions/insertions, including two 3-bp alterations. Sequencing of all 20 MIDIA-negative samples also showed highly consistent results with MIDIA because 10 cases were wild type and eight displayed a single base substitution in which detection by MIDIA was not expected. Sequencing also revealed a 3-bp deletion undetected by MIDIA, thus establishing the resolution limit of MIDIA at deletions/insertions >or=3 bp. Denaturing high-pressure liquid chromatography analysis confirmed all mutations detected by MIDIA and sequencing. We pro-pose MIDIA as the first step in mutational screening of GIST because it allowed the detection of 75% of mutated cases (94% of deletions/insertions) in less than 30 minutes after polymerase chain reaction amplification and at a lower cost compared with denaturing high-pressure liquid chromatography and sequencing, which might then be used only for MIDIA-negative cases.

Recent data from animal models has demonstrated that both endothelial and smooth muscle progenitor cells contribute to the development of atherosclerosis. However, no data exists concerning the presence of progenitor cells in human atherosclerotic vessels. In the present study, a range of normal and atherosclerotic human arteries were collected from patients undergoing coronary artery bypass surgery. Segments of internal mammary artery (normal controls), and segments of proximal ascending aorta with visible fatty streak were analysed. Immunofluorescence was used to detect a panel of progenitor cell markers. A small number of progenitor cells were identified within neointimal lesions and the adventitia with variable expression of CD34, stem cell antigen (Sca-1), c-kit and VEGF receptor 2 (VEGFR2) markers, but no CD133 expression. On average there was a two- to three-fold increase in progenitor cell number in the adventitia of atherosclerotic vessels compared with normal controls, with a significant difference (p<0.05) in the frequency of cells expressing VEGFR2. Thus, we have provided the first evidence that vascular progenitor cells exist within atherosclerotic lesions, and identified an increased number of progenitor cells in the adventitia of human atherosclerotic vessels. These cells might be a source for smooth muscle cells (SMCs), macrophages and endothelial cells (ECs) that form atherosclerotic lesions.

The expression of vimentin, alpha-smooth muscle actin (alpha-SMA) and c-kit in adenoid cystic carcinomas (AdCCs) and polymorphous low-grade adenocarcinomas (PLGAs) was investigated immunohistochemically to evaluate the application of these markers to distinguish AdCCs from PLGAs when the histological features are equivocal. Tissue specimens of AdCCs and of PLGAs, formalin-fixed and paraffin-embedded were retrospectively studied using vimentin, alpha-SMA and c-kit. Positive staining for alpha-SMA was identified in all AdCCs and 25% of PLGAs. The immunoreactivity of c-kit in all positive cases of AdCCs (83%) and PLGAs (41%) was more than 50% and less than 50% of tumor cells respectively. The expression pattern for both alpha-SMA and c-kit, in tubular structures of AdCCs was different of that seen in the same structures in PLGAs. The results of this study support the potential application of alpha-SMA and c-kit as an adjunctive aid in the differential diagnosis of AdCCs from PLGAs.

There is a great need in cell biology for the simultaneous detection of many intracellular and extracellular proteins within single cells. Current optical methods based on fluorescence activated flow cytometry are difficult to multiplex. We have developed a novel application of ICP-MS-linked metal-tagged immunophenotyping which has great potential for highly multiplexed proteomic analysis. Expression of intracellular oncogenic kinase BCR/Abl, myeloid cell surface antigen CD33, human stem cell factor receptor c-Kit and integrin receptor VLA-4 were investigated using model human leukemia cell lines. Antigens to which specific antibodies are available and are distinguishably tagged can be determined simultaneously, or multiplexed. Four commercially available tags (Au, Sm, Eu, and Tb) conjugated to secondary antibodies enable a 4-plex assay assuming that the primary antibodies are not cross-reactive. Results obtained by ICP-MS were compared with data from FACS. ICP-MS as an analytical detector possesses several advantages that enhance the performance of immunoassays, which are discussed in detail. Although multiplexing using metal-conjugated reagents is in a very early stage of research and feasibility studies, it is already apparent that more than four antigens could be accurately detected simultaneously using the ICP-MS instrument.

C-KIT (CD117), a tyrosine kinase receptor, is involved in the growth and development of normal tissues and some types of neoplasms. In the present study we analysed the expression of this molecule in salivary gland tumours. Archival formalin-fixed, paraffin-embedded sections of 40 benign and 57 malignant salivary gland tumours were retrieved and retrospectively studied immunohistochemically using a polyclonal C-KIT antibody in an Envision/HRP technique. In addition five samples of chronic submandibular sialadenitis, five normal minor salivary glands and parotid or submandibular gland tissue adjacent to benign tumour were also studied. C-KIT expression was observed in cases of adenoid cystic, acinic cell polymorphous low grade, epithelial-myoepithelial, carcinosarcoma and basal cell adenocarcinomas, as in luminal cells of pleomorphic adenomas, in serous acinar and only in intercalated and a small number of striated ductal cells of inflammatory salivary gland tissue, whereas normal salivary lobules were generally negative except a weak positivity of intercalated cells. Contrary to other reports, this study suggests that, C-KIT protein does not appear to be an exclusively specific marker for benign or malignant salivary gland neoplasms, but may be useful in differential diagnosis of adenoid cystic carcinoma from polymorphous low grade adenocarcinoma. Furthermore its expression in serous acinar cells in sialadenitis and intercalated ductal cells in normal and inflammatory lesions may indicate a possible participation in pathogenesis of both neoplastic and non-neoplastic salivary gland diseases.

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone metabolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for alpha-fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering.

Aim: The aim of this study is to evaluate the S100A1 and KIT as gene markers for the differentiation of common subtypes of renal tumours., Methods: Fifty-five tissue samples (15 clear cell RCCs, 15 papillary RCCs, 7 chromophobe RCCs, 8 oncocytomas and 10 normal renal tissues) were studied The gene expressions of S100A1 and KIT were analysed by one-step RT-PCR by using the specific primers., Results: S100A1 was expressed in 2/15 clear cell RCCs, 11/15 papillary RCCs, 7/8 oncocytomas and in 0/7 chromophobe RCCs. KIT gene was expressed in 6/7 chromophobe RCCs and 7/8 oncocytomas while 0/15 clear cell RCCs and 1/15 papillary RCCs expressed kit gene. Normal tissue expressed neither S100A1 nor KIT gene., Conclusion: S100A1 and KIT can be used as gene markers for the differentiation of common subtypes of renal tumours.

Gastrointestinal stromal tumour rarely develops in the duodenal ampulla region. We report here a case of gastrointestinal stromal tumour of the ampulla of Vater found in a 44-year-old Japanese man presenting with biliary obstruction. He died of hepatic failure with diffuse liver metastasis. The postmortem examination showed a large Borrman type III-like tumour in the duodenal ampullary region with direct invasion of the pancreas and extrahepatic bile duct as well as metastases to the liver and regional lymph nodes. The duct orifice was located at the centre of the tumour. Microscopically, the tumour consisted of anaplastic spindle cells with high mitotic activity (90 mitoses per 50 high-power fields). Immunohistochemically, the spindle cells were positive for KIT and CD34. The final diagnosis was high-grade malignant gastrointestinal stromal tumour of the ampulla of Vater. Considering the recent advances in the diagnosis and treatment of gastrointestinal stromal tumour, this neoplasm should be included in the differential diagnosis of the tumours appearing in the duodenal ampulla region.

Homozygous natural white-spotted (W) mutations in the gene encoding the receptor tyrosine kinase c-Kit are associated with hypoplastic bone marrow, severe macrocytic anemia, and lethality during early postnatal life. c-Kit(W/W) mice can be rescued by wild-type hematopoietic stem cells (HSCs), but it is not known whether the lethality of c-Kit(W/W) mice is the result of HSC failure or defects specific for erythropoiesis. Here we show that transgenic expression of erythropoietin (EPO) can overcome the lethality caused by the c-Kit(W/W) mutation. In W mutant mice rescued by EPO, termed WEPO, erythrocyte colony-forming units (CFU-Es) are rescued to normal frequencies. Hence, Epo receptor signals can partially bypass the strict requirement for c-Kit signaling in erythropoiesis in the absence of c-Kit in vivo. Using a series of W and rescue mouse strains, we define here the erythropoietic threshold permitting survival in vivo. The lethality of c-Kit(W/W) mice has precluded analysis of this crucial receptor-ligand pair in adult stem/progenitor cells. Our strategy to generate viable c-Kit(W/W) mice will be useful to analyze the role of this important receptor tyrosine kinase in adult life in vivo.

Most gastrointestinal stromal tumors (GISTs) carry activating mutations of the KIT gene encoding the receptor tyrosine kinase KIT. In a previous study we were able to show an association between the lack of KIT mutations (wild-type GISTs) and the presence of a significant epithelioid tumor component. A very recent study described the occurrence of PDGFRalpha mutations in KIT wt GISTS. Therefore, we studied a panel of 87 GISTs for mutations in the hot spot regions of the PDGFRalpha gene with single strand conformation polymorphism analysis and sequencing and correlated the PDGFRalpha status with pathomorphological data. We detected 20 cases with exon 18 mutations but none with exon 12 mutations. The mutations were located in the second kinase domain of PDGFRalpha with 16 point mutations, and four larger deletions of 9 to 12 bp. All cases with mutations in the PDGFRalpha gene revealed wild-type KIT in common regions of mutation, ie, exons 9 and 11. Most interestingly, the occurrence of PDGFRalpha mutations was significantly associated with a higher frequency of epithelioid or mixed morphology (18 of 20 cases, P < 0.0001) and gastric location (all cases, P = 0.0008). Our data indicate that GISTs represent distinctive entities, differing in genetic, biological, and morphological features.

This phase 2 pilot study was conducted to determine the efficacy and safety of imatinib mesylate in patients with c-kit-positive acute myeloid leukemia (AML) refractory to or not eligible for chemotherapy. Twenty-one patients were enrolled and received imatinib 600 mg orally once daily. Five responses were seen primarily in patients, starting with relatively low blast counts in bone marrow (BM) and peripheral blood (PB): 2 patients who were considered refractory on chemotherapy on the basis of persistence of blasts in PB and BM met the criteria for complete hematologic remission, 1 patient had no evidence of leukemia, and 2 patients achieved a minor response. Treatment with imatinib demonstrated a good safety profile and was well tolerated. Western blot analysis and immunohistochemistry demonstrated c-Kit activation in primary AML cells. Further, imatinib treatment of primary AML cells inhibited c-Kit tyrosine-phosphorylation. Genomic DNA-sequencing of c-KIT showed no mutations in exons 2, 8, 10, 11, 12, and 17. Although some of the responses derived from relatively small reductions in leukemic blasts and may be attributable, in part, to prior chemotherapy, these cases suggest that imatinib has interesting clinical activity in a subset of patients with c-kit-positive AML. Further clinical trials are warranted to explore the clinical potential of imatinib in AML and to identify the underlying molecular mechanism.

Lineage-marker depleted (Lin(-)) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin(-)Sca(+)Kit(+) or Lin(-)Sca(+)Kit(-) cells. Lin(-)Sca(+)Kit(-) cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP(3)), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.

Background: Recent studies have shown that several tumours express c-KIT, a growth factor receptor with tyrosine kinase activity; moreover, clinical results have shown the efficacy of the tyrosine kinase inhibitor, STI571, in c-KIT-positive tumours. The aim of this study was to determine the incidence and correlation with chemotherapy resistance of c-KIT expression in advanced serous, low grade of differentiation, ovarian carcinoma., Patients and Methods: We performed an immunohistochemistry analysis of 56 patients with advanced serous ovarian carcinomas using archival paraffin-embedded specimens., Results: Intense c-KIT immunostaining was observed in 51.7% of cases. c-KIT expression was statistically correlated with progression of disease after first-line chemotherapy (P = 0.029)., Conclusions: c-KIT is also expressed in ovarian carcinoma and it is statistically correlated with chemotherapy resistance. This study suggests the necessity for clinical trials confirming the utility of the tyrosine kinase inhibitor, STI571, in ovarian advanced cancer patients with c-KIT overexpression when these patients have shown no clinical response to conventional chemotherapy.

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.

Adult murine bone marrow hematopoietic stem cells (HSCs) can be purified by sorting Hoechst 33342-extruding side population (SP) cells. Herein we investigated whether SP cells reside within embryonic tissues and exhibit hematopoietic progenitor activity. We isolated yolk sac (YS) and embryonic tissues 7.5 to 11.5 days after coitus (dpc), resolved an SP in each, and demonstrated that these SP cells exhibit distinct phenotypic and functional characteristics throughout development. YS and embryonic SP isolated 8.0 dpc expressed vascular endothelial-cadherin (VE-cadherin) and vascular endothelial receptor 2 (Flk-1), markers not expressed by bone marrow SP but expressed by endothelial cells and progenitors. SP at this stage did not express CD45 or produce hematopoietic colonies in vitro. In contrast, SP isolated 9.5 to 11.5 dpc contained a significantly higher proportion of cells expressing cKit and CD45, markers highly expressed by bone marrow SP. Furthermore, YS SP isolated 9.5 to 11.5 dpc demonstrated 40- to 90-fold enrichment for hematopoietic progenitor activity over unfractionated tissue. Our data indicate that YS and embryonic SP cells detected prior to the onset of circulation express the highest levels of endothelial markers and do not generate blood cells in vitro; however, as development progresses, they acquire hematopoietic potential and phenotypic characteristics similar to those of bone marrow SP.

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP(+) KIT(-) fraction of prospermatogonia has much higher repopulating activity than does the GFP(+)KIT(+) population in the adult environment. Interestingly, the GFP(+)KIT(+) population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte-Sertoli cell adhesion and migration of gonoyctes toward the basement membrane.

Objective: Differentiating between adenoid cystic carcinomas (ACCs), polymorphous low-grade adenocarcinomas (PLGAs), and the monomorphic adenomas (including canalicular adenomas, trabecular adenomas, and basal cell adenomas) can present a diagnostic challenge, especially when examining tissue obtained from small incisional or fragmented biopsies. Recent studies have revealed that overexpression of the tyrosine kinase receptor protein c-kit occurs in a narrow subset of malignant neoplasms, including gastrointestinal stromal tumors, myeloid leukemias, seminomas, and ACCs. C-kit reportedly is not expressed in PLGAs. We compared the expression of the c-kit antigen in the malignant salivary gland neoplasms ACC and PLGA with its expression in salivary gland monomorphic adenoma (including canalicular adenoma and basal cell adenoma)., Study Design: Formalin-fixed paraffin-embedded sections of 49 salivary gland neoplasms (17 monomorphic adenomas, 17 PLGAs, and 15 ACCs) accessioned between 1989 and 2002 were retrieved from the files of the Department of Pathology, Long Island Jewish Medical Center, and were stained with an anti-c-kit polyclonal antibody., Results: C-kit reactivity was uniformly positive in the cytoplasm of luminal neoplastic cells in ACCs (15/15, 100%). Positive reactivity was also identified in the majority of PLGAs (16/17, 94%), with at least 25% of the tumor cells being positive. Similar reactivity was seen in monomorphic adenomas (16/17, 94%)., Conclusions: In contrast to previous reports, we find that c-kit expression was not restricted to ACC but was expressed in all 3 tumor types evaluated (ACC, PLGA, and monomorphic adenoma). Therefore, c-kit does not appear to be a useful marker for distinguishing between either ACC and PLGA in equivocal cases, or in benign and malignant salivary gland neoplasms.

The inability to undergo apoptosis is a crucial mechanism of multidrug resistance in acute myeloid leukemia (AML), and the analysis of mitochondrial apoptotic proteins may represent a significant prognostic tool to predict outcome. Bcl-2 and Bax oncoproteins were evaluated in 255 de novo AML patients (pts) by flow cytometry using an anti-bcl-2 monoclonal antibody (MoAb) and an anti-bax MoAb. The results were expressed as an index (bax/bcl-2) obtained by dividing bax mean fluorescence intensity (MFI) and bcl-2 MFI. Lower bax/bcl-2 ratio was associated with French-American-British (FAB) M0-M1 classes (P =.000 01) and CD34 more than 20% (P <.000 01). There were striking inverse correlations between CD34 or CD117 MFI and bax/bcl-2 values (r = -.40, P <.000 001 and r = -.29, P =.000 002), confirming that immaturity is consistent with this index. Moreover, lower bax/bcl-2 levels were correlated with poor-risk cytogenetics (P =.0002). A significant higher complete remission (CR) rate was found in pts with higher bax/bcl-2 levels (79% versus 45%; P =.000 01). Also, both a longer overall survival (OS) and disease-free survival (DFS) were observed in pts with higher bax/bcl-2 levels (P =.000 01 and =.019). Noteworthy, bax/bcl-2 levels accurately predicted the clinical response and outcome of pts with normal or unknown cytogenetics. Indeed, within this subset of 147 pts, higher bax/bcl-2 ratio was significantly associated both with a higher CR rate (86% versus 42%; P <.000 01) and a longer OS (P =.0016). The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis. Therefore, mitochondrial oncoproteins, such as bcl-2 and bax, represent both sensitive indicators of clinical outcome and potential targets of novel proapoptotic molecules in order to circumvent chemoresistance.

It was recently reported that crude bone marrow cells have the ability to differentiate into glomerular mesangial cells. However, the exact nature of the engrafting cells in the bone marrow was not known. We tested the hypothesis that hematopoietic stem cells are capable of reconstituting the mesangial cells by transplanting a clonal population of cells derived from a single stem cell. We cultured Lin(-), Sca-1(+), c-kit(+), CD34(-) bone marrow cells from transgenic enhanced green fluorescent protein (EGFP) mice (C57BL/6-Ly-5.2 background) individually for 1 week in the presence of interleukin-11 and steel factor. We then transplanted viable clones individually into lethally irradiated C57BL/6-Ly-5.1 mice. Kidneys from 5 recipient mice showing high levels (60%-90%) of multilineage hematopoietic reconstitution were examined 2 to 6 months later, using differential interference contrast and epifluorescence microscopy. EGFP(+) cells with a morphology characteristic of mesangial cells were evident within the glomeruli. Transplantation of 100 noncultured Lin(-), Sca-1(+), c-kit(+), CD34(-) bone marrow cells also generated mesangial cells. Cultured EGFP(+) glomerular cells from recipient mice contracted in response to angiotensin II. EGFP(+) mesangial cells seen in male-to-male transplants revealed only one Y-chromosome. These data demonstrate that a single hematopoietic stem cell is capable of differentiating into glomerular mesangial cells and that the process does not involve cell fusion.

The role of thrombopoietin (Tpo) in promoting hematopoiesis has been extensively studied in late fetal, neonatal, and adult mice. However, the effects of Tpo on early yolk sac hematopoiesis have been largely unexplored. We examined whole embryos or the cells isolated from embryo proper and yolk sacs and identified both Tpo and c-mpl (Tpo receptor) mRNA transcripts in tissues as early as embryonic day 6.5 (E6.5). Presomite whole embryos and somite-staged yolk sac and embryo proper cells were plated in methylcellulose cultures and treated with selected hematopoietic growth factors in the presence or absence of Tpo. Tpo alone failed to promote colony-forming unit (CFU) formation. However, in the presence of other growth factors, Tpo caused a substantial dose-dependent reduction in primitive and definitive erythroid CFU growth in cultures containing E7.5 and E8.0 whole embryos and E8.25 to 9.5 yolk sac-derived cells. Meanwhile, Tpo treatment resulted in a substantial dose-dependent increase in CFU-mixed lineage (CFU-Mix) and CFU-megakaryocyte (CFU-Meg) formation in cultures containing cells from similar staged tissues. Addition of Tpo to cultures of sorted E9.5 yolk sac c-Kit(+)CD34(+) hematopoietic progenitors also inhibited erythroid CFU growth but augmented CFU-Mix and CFU-Meg activity. Effects of Tpo on CFU growth were blocked in the presence of a monoclonal antibody with Tpo-neutralizing activity but not with control antibody. Thus, under certain growth factor conditions, Tpo directly inhibits early yolk sac erythroid CFU growth but facilitates megakaryocyte and mixed lineage colony formation.

Recently some reports have suggested that gastrointestinal stromal tumors (GIST) might originate from the interstitial cells of Cajal or differentiate into them because they express c-kit and/or CD34 and indicated that the majority of previously diagnosed smooth muscle tumors (SMT) actually belong to GIST, but are not true SMT. We, therefore, detected c-kit, CD34, SMA, and S-100 in 106 Chinese cases of gastrointestinal tumors, which were histopathologically diagnosed as smooth muscle tumors originally, to demonstrate the immunophenotypes of these tumors. The results showed that 73 cases had immunoreaction with c-kit and/or CD34, of which 48 cases showed coexpression with either SMA or S-100 or with both. A correlation between the immunophenotypes and known histopathological parameters was also shown here based on follow-up data. We suggest that the concept of GIST should not be used as an umbrella to cover all gastrointestinal mesenchymal tumors, but be defined in a narrow term as differing from true smooth muscle tumors., (Copyright 2002 Elsevier Science (USA).)