Stem Cell Factor Receptor C-kit Research Articles

Stem cell factor/c-kit signaling mediated cardiac stem cell migration via activation of p38 MAPK

It was reported that there are cardiac stem cells (CSCs) in the rat heart, and they could reconstitute well-differentiated myocardium that are formed by blood-carrying new vessels and myocytes. However, how do the CSCs migrate into the peri-infarcted areas after myocardial infarction (MI)? It remains entirely unknown about the signal transduction involved in the migration of CSCs. Rat heart MI was induced by left coronary artery ligation. Both immunohistochemical staining and Western blotting analysis was performed to detect the expression of SCF protein, and RT-PCR was conducted for the expression of SCF mRNA. Cardiac stem cells were isolated from rat hearts, and a cardiac stem cell migration assay was performed using a 48-well chemotaxis chamber system. On day 5 after MI in rats, the expression of stem cell factor (SCF) mRNA and protein was significantly increased in the peri-infarcted area, which was matched with more accumulation of CSCs in the region and improvement of cardiac function, which was blocked by p38 MAPK selective inhibitor SB203580. In in vitro experiments, SCF induced CSC migration in a concentration-dependent manner, and the antibody against SCF receptor (c-kit) blocked the SCF-induced CSC migration. Western blot analysis showed that the phosphorylated p38 MAPK (Phospho-p38 MAPK) was highly increased in the SCF-treated CSCs, and the inhibition of p38 MAPK activity significantly attenuated SCF-induced the migration of CSCs. It demonstrated that SCF/c-kit signaling may mediate the migration of CSCs via activation of p38 MAPK.

The Kinase Inhibitors Sunitinib (Sutent®) and Sorafenib (Nexavar®) Differentially Affect Reactivity of NK Cells Against Renal Cell Cancer.

Sunitinib (Sutent®) and Sorafenib (Nexavar®) are multikinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) because of their direct and indirect anti-tumor and anti-angiogenic effects. Sunitinib and Sorafenib target, among others, vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), the stem cell factor receptor (cKIT) and Fms-like tyrosine kinase-3 (Flt3). Beyond that, Sorafenib additionally inhibits c-RAF and B-RAF. NK cells play an important role in the immune surveillance of tumors. Their reactivity is governed by different surface receptors leading to activation of various kinases, phosphatases and transcription factors. Up to now, nothing is known regarding the influence of Sunitinib and Sorafenib on NK cells. Here we report that Sunitinib and Sorafenib differentially affect NK cell cytotoxicity. Analysis of peripheral blood mononuclear cells from 10 independent donors revealed that Sorafenib but not Sunitinib significantly (p<0.05, Mann-Whitney U-test) reduced cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib significantly (p<0.05, Mann-Whitney U-test) diminished cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI-2 (from 46% to 14%, from 52% to 22% and from 63% to 42%, respectively) in chromium release assays. Again, no inhibition of cytotoxicity was observed in the presence of pharmacological concentrations of Sunitinib. Since activation of MAP kinases leading to ERK phosphorylation regulates the movement of lytic granula towards target cells, which is responsible for NK cell cytotoxicity we investigated the influence of Sunitinib and Sorafenib on ERK phosphorylation. While Sunitinib had no effect, Sorafenib, alike the specific c-RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation as determined by Western Blot. Our results indicate that NK cell anti-tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.

Functional Interaction of c-Kit Receptor and Interleukin-3 Receptor.

Hematopoiesis is regulated by a number of growth factors and cytokines, among which stem cell factor (SCF) plays a critical role for normal hematopoiesis. An important feature of SCF is its strong capacity to synergize with the cytokines including interleukin-3(IL-3), granulocyte-marcrophage colony-stimulation factor (GM-CSF) and erythropoietin to regulate proliferation and differentiations of hematopoietic progenitor cells. The mouse hematopoietic progenitor cell line EML contains two populations of lineage negative cells, distinguished by the expression or absence of CD34. The two sub-populations express similar levels of the SCF receptor c-kit, but only the CD34+ population replicates in the presence of SCF alone, while the CD34 negative population replicates in the presence of IL3 but this replication is synergistically stimulated by the addition of SCF. Our previous studies indicated that synergistic proliferation of the mouse hematopoietic progenitor cell line EML induced by SCF in combination with IL-3 was probably mediated by the synergistic increases in tyrosine phosphorylation of c-Kit and β-chain of IL-3 receptor, which is shared with GM-CSF receptor and interleukin-5 receptor. Conversely, the decreased response of c-kit to SCF alone in the CD34 negative population may be a result of the increased expression of IL3 receptor in these cells. Trans-phosphorylation between c-Kit and the β-chain of IL-3 receptor was also observed in EML cells. In studies of the molecular mechanism behind the functional interactions between SCF and IL-3 we found that c-Kit and IL-3 receptor β-chain form a complex in EML cells. Antibody Ab-1 specific against the Ig-like domain 4 in the extracellular region of c-Kit blocked not only cell proliferation induced by SCF but also synergistic proliferation induced by SCF plus IL-3. Consistent with this finding, Ab-1 inhibited phosphorylation of c-Kit induced by SCF or IL-3, and synergistic phosphorylation of c-kit induced by SCF plus IL-3 respectively. SCF and IL-3 in combination synergistically activated the protein kinases Akt and Erks, the downstream mediators of c-Kit and IL-3R. Phosphorylation of Erks is independent on the phosphatidylinositol 3-kinase (PI3-kinase) pathway in EML cells. These data suggested the possibility that the synergistic growth of EML cells induced by SCF in combination with IL-3 was mediated by multiple mechanisms that could include trans- and synergistic phosphorylation of c-Kit and the β-chain of IL-3 receptor, PI3-kinase dependent synergistic phosphorylation of Akt and PI3-kinase independent synergistic phosphorylation of Erks. The trans- and synergistic phosphorylation of c-Kit and β-chain of IL-3 receptor was mediated by physical interaction of two receptors.

Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington's disease

Neural and mesenchymal stem cells have been proposed as alternative sources of cells for transplantation into the brain in neurodegenerative disorders. However, the endogenous factors controlling their engraftment within the injured parenchyma remain ill-defined. Here, we demonstrate significant engraftment of undifferentiated exogenous mesenchymal or neural stem cells throughout the lesioned area in a rat model for Huntington's disease, as late as 8 weeks post-transplantation. We show that stem cell factor (SCF), strongly up-regulated within host cells in the damaged striatum, is able to activate the SCF receptor c-kit and its signaling pathway and to promote the migration and proliferation of mesenchymal and neural stem cells in vitro. Furthermore, c-kit receptor blockade alters neural stem cell distribution within the lesioned striatum. Host SCF expression is observed in atypical cells expressing glial fibrillary acidic protein and doublecortin in the lesioned striatum and in migrating doublecortin-positive progenitors. Taken together, these data demonstrate that SCF produced in situ in the lesioned striatum is an important factor in promoting the engraftment of stem cells within the lesioned brain.

Why human extragonadal germ cell tumours occur in the midline of the body: old concepts, new perspectives

Hypotheses on the origin and distribution of extragonadal germ cell tumours (GCTs) and teratomas are briefly reviewed and revisited in the light of (i) new developments in the classification of GCTs, (ii) data on genomic imprinting of these neoplasms and (iii) the recent finding that germ cells can be derived from mouse and human embryonal stem (ES) cells. Only the Type I (infantile teratomas/yolk sac tumours) and Type II GCTs (seminomatous tumours and non-seminomas) occur in the gonads and extragonadal localizations. The data on genomic imprinting lend support to the hypothesis that they are derived from germ cells. These precursor cells could have differentiated from ES cells in extragonadal localizations. Their distribution along the midline of the body is still best explained by the migration of primitive germ cells during development. The narrower distribution of the Type II than the Type I GCTs is probably due to the more strict conditions for survival and proliferation of primordial germ cells (PGCs)/gonocytes from which the Type II tumours originate, when compared with the precursor cells of Type I tumours, probably primitive germ cells closer to the ES cell. The known niches in which the Type II tumours develop have in common that they contain feeder cells expressing stem cell factor (SCF) - the ligand for the SCF receptor c-KIT, involved in proliferation and survival of PGCs/gonocytes - and contain GBY including the gene TSPY.

IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development

We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit+), stem cell antigen-1 (sca-1+), hematopoietic stem cell (HSC), or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions.

Crystal Structure of the Tyrosine Kinase Domain of Colony-stimulating Factor-1 Receptor (cFMS) in Complex with Two Inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

Everolimus Is Comparably Effective to Rapamycin Acting Synergistically with Imatinib in the Treatment of Constitutively Active Mutations of the Juxtamembrane Domain of c-Kit.

Kit cITD is a complex internal tandem duplication involving the juxtamembrane (JMD) and kinase domain of stem cell factor receptor c-kit which was identified recently in 7% of childhood AML. kit cITD induces constitutive proliferation and apoptosis resistance that can be synergistically blocked with imatinib (im) and rapamycin (ra).Due to a long half-life ra has disadvantageous pharmacological characteristics making drug handling especially difficult in children with serum concentrations quickly raising to toxic levels. We therefore compared ra with its ester analogue everolimus (RAD001, ev) which has a better bioavailability and a shorter half-life in order to assess equivalence and potential functional superiority. Ba/F3 cells, stably transfected with c-kit WT and kit cITD, were treated with im, ra and ev alone and in combination. In proliferation assays of WT and cITD cell lines ev was a significantly more potent inhibitor than ra. When ra or ev were combined with low im concentrations proliferation was synergistically inhibited with both drugs but again significantly better with ev. In cITD expressing cells ra and ev alone were comparably poor inducers of apoptosis. In assays combining im with ra or ev both combinations showed a significant and comparable synergism with an additional dose effect of the mTOR inhibitors between 3nM and 0.3 nM. In a NOD/SCID animal model all animals injected with kit cITD succumbed due to multi-organ infiltration of c-kit positive cells within 20 days. Survival was prolonged in animals treated with im/ra and ev. We therefore conclude that ev is at least equivalent to ra and can replace ra exploring a molecular based therapeutic attempt in susceptible children with identified mutations of the JMD of c-kit.

A c-fms tyrosine kinase inhibitor, Ki20227, suppresses osteoclast differentiation and osteolytic bone destruction in a bone metastasis model

In bone metastatic lesions, osteoclasts play a key role in the development of osteolysis. Previous studies have shown that macrophage colony-stimulating factor (M-CSF) is important for the differentiation of osteoclasts. In this study, we investigated whether an inhibitor of M-CSF receptor (c-Fms) suppresses osteoclast-dependent osteolysis in bone metastatic lesions. We developed small molecule inhibitors against ligand-dependent phosphorylation of c-Fms and examined the effects of these compounds on osteolytic bone destruction in a bone metastasis model. We discovered a novel quinoline-urea derivative, Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea), which is a c-Fms tyrosine kinase inhibitor. The IC(50)s of Ki20227 to inhibit c-Fms, vascular endothelial growth factor receptor-2 (KDR), stem cell factor receptor (c-Kit), and platelet-derived growth factor receptor beta were found to be 2, 12, 451, and 217 nmol/L, respectively. Ki20227 did not inhibit other kinases tested, such as fms-like tyrosine kinase-3, epidermal growth factor receptor, or c-Src (c-src proto-oncogene product). Ki20227 was also found to inhibit the M-CSF-dependent growth of M-NFS-60 cells but not the M-CSF-independent growth of A375 human melanoma cells in vitro. Furthermore, in an osteoclast-like cell formation assay using mouse bone marrow cells, Ki20227 inhibited the development of tartrate-resistant acid phosphatase-positive osteoclast-like cells in a dose-dependent manner. In in vivo studies, oral administration of Ki20227 suppressed osteoclast-like cell accumulation and bone resorption induced by metastatic tumor cells in nude rats following intracardiac injection of A375 cells. Moreover, Ki20227 decreased the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells on bone surfaces in ovariectomized (ovx) rats. These findings suggest that Ki20227 inhibits osteolytic bone destruction through the suppression of M-CSF-induced osteoclast accumulation in vivo. Therefore, Ki20227 may be a useful therapeutic agent for osteolytic disease associated with bone metastasis and other bone diseases.

Effects of PKC412, Nilotinib, and Imatinib Against GIST-Associated PDGFRA Mutants With Differential Imatinib Sensitivity

Activating mutations in platelet-derived growth factor receptor alpha (PDGFRA) have been reported in a subset of gastrointestinal stromal tumor (GIST) patients who do not express the mutant stem cell factor receptor c-kit. The responsiveness of mutant PDGFRA-positive GIST to imatinib depends on the location of the PDGFRA mutation; for example, the V561D juxtamembrane domain mutation is more sensitive to imatinib than the D842V kinase domain mutation. In this study, we compare the effects of 3 tyrosine kinase inhibitors, PKC412 and nilotinib, and imatinib, on 2 GIST-related PDGFRA mutants, V561D and D842V, which possess differential sensitivity to imatinib. The effects of PKC412, nilotinib, and imatinib, alone and in combination, were evaluated via in vitro proliferation studies performed with V561D- or D842V-PDGFRA mutants. The effects of nilotinib and PKC412, alone and combined, were investigated in vivo. PKC412 potently inhibited the V561D-PDGFRA mutant in vitro and the D842V-PDGFRA mutant in vitro and in vivo. Both imatinib and nilotinib displayed potent activity in vitro against the V561D-PDGFRA mutant but were significantly less efficacious against D842V-PDGFRA. However, when combined with either imatinib or PKC412, nilotinib showed no evidence for antagonism and acted in a cooperative fashion against D842V-PDGFRA. Our findings support the clinical testing of PKC412 for treatment of mutant PDGFRA-GIST. The data also support the use of nilotinib as a treatment option for V561D-PDGFRA-associated GIST, although the reduced sensitivity of D842V-PDGFRA probably limits the potential of nilotinib monotherapy for D842V-PDGFRA-associated GIST.

Lack of evidence that bone marrow cells contribute to cholangiocyte repopulation during experimental cholestatic ductal hyperplasia

Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear. Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated. Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7. In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.

Unilateral induction of progenitors in the spinal cord of hSOD1 G93A transgenic rats correlates with an asymmetrical hind limb paralysis

Transgenic rats expressing a mutated form of the human Cu/Zn superoxide dismutase (hSOD1 G93A) develop an amyotrophic lateral sclerosis (ALS)-like phenotype, including motor neurone degeneration and reactive gliosis in the spinal cord. This study aimed at examining the presence of endogenous neural progenitors in the lumbar spinal cord of these rats at the end-stage of the disease. Immunohistochemical data clearly demonstrated the induced expression of the stem cell factor reported as a chemoattractant and survival factor for neural stem cells as well as nestin (neuro-epithelial stem cell intermediate filament) in the spinal cord sections. While the stem cell factor immunolabelling appeared diffuse throughout the gray matter, nestin labelling was restricted to clusters within the ventral horn. Interestingly, as paralysis regularly develops asymmetrically, induction of nestin was only detected on the ipsilateral side of the predominant symptoms. Finally, immunohistochemical detection of the stem cell factor receptor (c-Kit) revealed its specific induction which coincided with nestin immunolabelling. Together, these results are indicative of endogenous recruitment of neural progenitors within lesioned tissues and could support the development of treatments involving endogenous or exogenous stem cells.

Pulmonary vein stenosis: expression of receptor tyrosine kinases by lesional cells

Background Primary pulmonary vein stenosis (PVS) is a progressive disorder of infants. Although catheter based intervention and chemotherapy are used to manage the disorder, the benefit of these approaches is reduced considerably by restenosis. The nature of the intimal cells causing the occlusive lesions in PVS is poorly understood. Methods Seven PVS cases were studied with antibodies for smooth muscle actin (SMA), muscle-specific actin (MSA), monoclonal desmin, S100 protein, CD31, CD34, CD45RO, CD68, CD99, Ki-67 (MIB-I), and with antibodies directed against several receptor tyrosine kinases (RTK), including platelet-derived growth factor alpha and beta receptor (PDGFR-α and -β), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor 1 and 2 receptor (VEGFR), and stem cell factor receptor (c-kit). Results Lesional cells stained strongly and diffusely with SMA and MSA, but not for macrophage, lymphocyte, endothelial markers, or for Ki-67. RTK expression was strong and diffuse for PDGFR-α and -β, FGFR, and VEGFR-2. Lesional cells stained for VEGF and PDGF β receptor was phosphorylated. Conclusions The histologic appearance, and the strong diffuse immunoreactivity for smooth muscle markers, indicates that the intimal lesional cells are myofibroblast-like. Expression of various receptor tyrosine kinases and some ligands suggests an autocrine or paracrine role of these proteins in the pathogenesis of the intimal occlusive lesion in PVS.

Two types of precursor cells in a multipotential hematopoietic cell line.

The biochemistry of early stages of hematopoietic differentiation is difficult to study because only relatively small numbers of precursor cells are available. The murine EML cell line is a multipotential cell line that can be used to model some of these steps. We found that the lineage- EML precursor cells can be separated into two populations based on cell surface markers including CD34. Both populations contain similar levels of stem cell factor (SCF) receptor (c-Kit) but only the CD34+ population shows a growth response when treated with SCF. Conversely, the CD34- population will grow in the presence of the cytokine IL-3. The human beta-globin locus control region hypersensitive site 2 plays different roles on beta-globin transcription in the CD34+ and CD34- populations. The two populations are present in about equal amounts in culture, and the CD34+ population rapidly regenerates the mixed population when grown in the presence of SCF. We suggest that this system may mimic a normal developmental transition in hematopoiesis.

Multiple cellular antigen detection by ICP-MS

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.

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.

Peritransplant Ischemic Injury Is Associated With Up-Regulation of Stromal Cell-Derived Factor-1

We evaluated chimerism and stromal cell-derived factor-1 (SDF-1) expression in response to peritransplant ischemic injury following human heart transplantation. Myocardial ischemia has been shown to trigger mobilization of stem cells to the heart in animal experiments. Between January 1998 and April 2002, a total of 114 male recipients received hearts from female donors. Of these 114 recipients, 26 had evidence of ischemic injury on their initial heart biopsies (ischemia group). These were compared to the remaining 88 patients (control group). Heart biopsy specimens obtained initially at one week and at one year after transplant were evaluated from 20 matched patients of each group for the presence of Y chromosome-containing nuclei. The SDF-1 messenger ribonucleic acid (mRNA) and protein expression were also evaluated on initial heart biopsy specimens. At one week, Y chromosome-containing nuclei were significantly increased in the ischemia group (0.68% vs. 0.04%; p < 0.0001) compared to the control group. These were positive for the stem cell factor receptor c-kit. A significant 3.3-fold increased mRNA expression (p = 0.001) and 2.8-fold increased protein expression (p = 0.01) of SDF-1 was noted in the ischemia group. At one year, Y chromosome was detected in 0.29% of cardiomyocyte nuclei in the ischemia group but none in the control group. The ischemia group had poorer survival and increased vasculopathy. This is the first report to describe chimerism and up-regulation of SDF-1 in human heart transplantation in response to ischemic injury.

Correlation of Murine Embryonic Stem Cell Gene Expression Profiles with Functional Measures of Pluripotency

Global gene expression profiling was performed on murine embryonic stem cells (ESCs) induced to differentiate by removal of leukemia inhibitory factor (LIF) to identify genes whose change in expression correlates with loss of pluripotency. To identify appropriate time points for the gene expression analysis, the dynamics of loss of pluripotency were investigated using three functional assays: chimeric mouse formation, embryoid body generation, and colony-forming ability. A rapid loss of pluripotency was detected within 24 hours, with very low residual activity in all assays by 72 hours. Gene expression profiles of undifferentiated ESCs and ESCs cultured for 18 and 72 hours in the absence of LIF were determined using the Affymetrix GeneChip U74v2. In total, 473 genes were identified as significantly differentially expressed, with approximately one third having unknown biological function. Among the 275 genes whose expression decreased with ESC differentiation were several factors previously identified as important for, or markers of, ESC pluripotency, including Stat3, Rex1, Sox2, Gbx2, and Bmp4. A significant number of the decreased genes also overlap with previously published mouse and human ESC data. Furthermore, several membrane proteins were among the 48 decreased genes correlating most closely with the functional assays, including the stem cell factor receptor c-Kit. Through identification of genes whose expression closely follows functional properties of ESCs during early differentiation, this study lays the foundation for further elucidating the molecular mechanisms regulating the maintenance of ESC pluripotency and facilitates the identification of more reliable molecular markers of the undifferentiated state.

Differentiation and development of human female germ cells during prenatal gonadogenesis: an immunohistochemical study

In the development of the human ovary, the second trimester includes the transition from oogonial replication to primordial follicle formation. The present study was carried out to assess differentiation and proliferation of germ cells in a series of female gonads from 19 fetuses from the second and third trimester, and two neonates. Using immunohistochemistry, the following markers were studied: placental/germ-like cell alkaline phosphatases (PLAP), the marker of pluripotency OCT3/4, the proliferation marker Ki-67, beta-catenin and E-cadherin, the stem cell factor receptor c-KIT, and VASA, a protein specific for the germ cell lineage. PLAP and OCT3/4 were seen during oogenesis, but not in germ cells engaged in folliculogenesis. A similar pattern was observed for Ki-67. Loss of pluripotency occurs once oocytes engage in follicle formation, suggesting a role of cell-cell interactions in the process of germ cell maturation. VASA, c-KIT, beta-catenin and E-cadherin were found in germ cells at all developmental stages of oogenesis and folliculogenesis. Immunohistochemically, two groups of germ cells can be distinguished. Germ cells that are predominantly found in the cortical region of the ovary before weeks 22-24 of gestation, showing an immature phenotype, are mitotically active, and express OCT3/4, a marker of pluripotency. On the other hand, germ cells undergoing folliculogenesis have lost their pluripotent potential and no longer proliferate.

The Selective Class III/V Receptor Tyrosine Kinase Inhibitor SU11657 Inhibits Tumor Growth and Angiogenesis in Experimental Neuroblastomas Grown in Mice

Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. SU11657 (SUGEN) is a selective multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activity exerted by targeting PDGF receptors (PDGFR), VEGF receptors (VEGFR), stem cell factor receptor (c-KIT), and FMS-related tyrosine kinase 3. Oral administration of SU11657 at 40 mg x kg(-1) x d(-1) to athymic mice resulted in significant growth inhibition of a panel of s.c. human neuroblastoma xenografts, namely, fast-growing SK-N-AS, MYCN- amplified IMR-32, and SH-SY5Y, by 90, 93.8, and 88%, respectively, and was well tolerated. All of the cell lines expressed VEGFR-2, PDGFR-beta, and c-KIT protein in the tumor cell and endothelial cell compartment by immunohistochemistry, and the expression decreased during therapy. Plasma concentrations of VEGF-A, PDGF-BB, and stem cell factor increased per milliliter of tumor volume at days 10, 18, and 20 of therapy. Furthermore, SU11657 reduced tumor angiogenesis by 63-96%. Our experimental data suggest that the angiogenesis inhibitor SU11657 may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood, such as neuroblastoma. In summary, the class III/V receptor tyrosine kinases and their ligands are implicated in angiogenesis, tumor cell proliferation, and cell survival, and it seems reasonable to determine whether interference with these pathways can suppress neuroblastoma growth or not.