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OVERVIEW: We show the existence of adult human mesenchymal progenitor cells (hMPCs) that can proliferate,in a cytokine-dependent manner,as individual cells in stirred suspension cultures (SSC) while maintaining their ability to form functional differentiated mesenchymal cell types. MATERIALS AND METHODS: Ficolled human bone marrow (BM)-derived cells were grown in SSC (and adherent controls) in the presence and absence of exogenously added cytokines. Phenotypic,gene expression,and functional assays for hematopoietic and nonhematopoietic cell populations were used to kinetically track cell production. Limiting-dilution analysis was used to relate culture-produced cells to input cell populations. RESULTS: Cytokine cocktail influenced total and progenitor cell expansion,as well as the types of cells generated upon plating. Flow cytometric analysis of CD117,CD123,and CD45 expression showed that cytokine supplementation influenced SSC output. The concomitant growth of CD45(+) and CD45(-) cells in the cultures that exhibited the greatest hMPC expansions suggests that the growth of these cells may benefit from interactions with hematopoietic cells. Functional assays demonstrated that the SSC-derived cells (input CFU-O number: 1990+/-377) grown in the presence of SCF+IL-3 resulted,after 21 days,in the generation of a significantly greater number (ptextless0.05) of bone progenitors (33,700+/-8763 CFU-O) than similarly initiated adherent cultures (214+/-75 CFU-O). RT-PCR analysis confirmed that the SSC-derived cells grown in osteogenic conditions express bone-specific genes (Cbfa1/Runx2,bone sialoprotein,and osteocalcin). CONCLUSIONS: Our approach not only provides an alternative strategy to expand adult BM-derived nonhematopoietic progenitor cell numbers in a scalable and controllable bioprocess,but also questions established biological paradigms concerning the properties of connective-tissue stem and progenitor cells. View Publication

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study,it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and,unexpectedly,to skeletal muscle cells in mice. In fact,the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions,CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells,thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org. View Publication

Pyrazole-based inhibitors of the transforming growth factor-beta type I receptor kinase domain (TbetaR-I) are described. Examination of the SAR in both enzyme- and cell-based in vitro assays resulted in the emergence of two subseries featuring differing selectivity versus p38 MAP kinase. A common binding mode at the active site has been established by successful cocrystallization and X-ray analysis of potent inhibitors with the TbetaR-I receptor kinase domain. View Publication

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV,saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV,almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF,whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1,Q2,and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV,-18.6 +/- 5.8 mV,and -15.2 +/- 2.7 mV,respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied,the Q1 vs. V curve was not significantly shifted,but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation,consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist,R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K. View Publication

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha,beta and gamma. Since RAR gamma is predominantly expressed in adult skin,specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay,we have identified three compounds with high RAR gamma selectivity. View Publication

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in textgreater95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P textless 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P textless 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture,33% and 72% of hematopoietic cells survived IR- and BU-induced damage,respectively,as compared with control cells,but they could not form colony forming units-granulocyte macrophages. Moreover,these surviving cells expressed an increased level of senescence-associated beta-galactosidase,p16(Ink4a),and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis,whereas BU does so mainly by inducing premature senescence. In addition,induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly,the induction of hematopoietic cell senescence by IR,but not by BU,was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner,whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway. View Publication

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer,and in particular,unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties,we have begun to target EphA2 on tumor cells using agonistic antibodies,which mimic the consequences of ligand binding. In our present study,we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells,which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand,as well as this subset of EphA2 antibodies. Finally,we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together,these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells. View Publication

Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here,we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10,25,or 50 mWcm(-2) and,(1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells,the photobleaching,and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However,in all of the cases,cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT,as well as confirming the likely role of (1)O(2) in porphyrin-based PDT. View Publication

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone,increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the side population" (SP) of marrow stem cells� View Publication

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However,the molecular mechanisms that regulate their differentiation,proliferation,and endothelial sheet formation remain to be elucidated. Here,we show that members of the transforming growth factor (TGF)-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5-days postcoitum embryos. TGF-beta and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly,SB-431542,a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin,facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover,SB-431542 up-regulated the expression of claudin-5,an endothelial specific component of tight junctions. These results suggest that endogenous TGF-beta/activin signals play important roles in regulating vascular growth and permeability. View Publication

In this study,we examined the effects of isoform-specific functional inhibitors of lysophosphatidic acid acyltransferase (LPAAT),which converts lysophosphatidic acid to phosphatidic acid,on multiple myeloma (MM) cell growth and survival. The LPAAT-beta inhibitors CT-32176,CT-32458,and CT-32615 induced textgreater95% growth inhibition (P textless 0.01) in MM.1S,U266,and RPMI8226 MM cell lines,as well as MM cells from patients (IC(50),50-200 nM). We further characterized this LPAAT-beta inhibitory effect using CT-32615,the most potent inhibitor of MM cell growth. CT-32615 triggered apoptosis in MM cells via caspase-8,caspase-3,caspase-7,and poly (ADP-ribose) polymerase cleavage. Neither interleukin 6 nor insulin-like growth factor I inhibited CT-32615-induced apoptosis. Dexamethasone and immunomodulatory derivatives of thalidomide (IMiDs),but not proteasome inhibitor PS-341,augmented MM cell apoptosis triggered by LPAAT-beta inhibitors. CT-32615-induced apoptosis was associated with phosphorylation of p53 and c-Jun NH(2)-terminal kinase (JNK); conversely,JNK inhibitor SP600125 and dominant-negative JNK inhibited CT-32615-induced apoptosis. Importantly,CT-32615 inhibited tumor necrosis factor-alpha-triggered nuclear factor-kappaB activation but did not affect either tumor necrosis factor-alpha-induced p38 mitogen-activated protein kinase phosphorylation or interleukin 6-triggered signal transducers and activators of transcription 3 phosphorylation. Finally,although binding of MM cells to bone marrow stromal cells augments MM cell growth and protects against dexamethasone-induced apoptosis,CT-32615 induced apoptosis even of adherent MM cells. Our data therefore demonstrate for the first time that inhibiting LPAAT-beta induces cytotoxicity in MM cells in the bone marrow milieu,providing the framework for clinical trials of these novel agents in MM. View Publication