技术资料

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 microM. The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells,indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells,suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary,cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells,indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation. View Publication

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture,differentiation of embryonic or simple epithelial cells occurred in both ES cell populations,as detected by expression of keratins 8,18,and 19. From 21 days,expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast,keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface,consistent with the association of these a subunits with beta 1. Furthermore,alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro,they did form keratinocyte cysts expressing alpha 6 beta 4,keratins 1 and 14,and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore,beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy,extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures,but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane. View Publication

To understand the mechanism of the sequential restriction of multipotency of stem cells during development,we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium,the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures. View Publication

We describe a new procedure for large-scale CB processing in the collection bag,thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C,the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal,the cell pellet was resuspended and the percent recovery of total WBC,CD34+ progenitor cells,CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300,600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC,CFU,CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy,effective and particularly suitable for large-scale banking under GMP conditions. View Publication

Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2),IL-7,and IL-15 cytokines,which share gamma c receptor subunit,in NK cell differentiation,we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF),IL-2,and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells,while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors,it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL-15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients. View Publication

In the past,the analysis of primitive human hematopoietic progenitor cells with repopulating activity was limited by lack of appropriate in vitro assay systems. It was recently shown that cobblestone area-forming cells (CAFC) giving rise to cobblestone areas after 5 weeks in long-term marrow cultures (LTMC) represent a population of pluripotent progenitor cells with long-term marrow-repopulating activity. We have used a microtiter limiting dilution-type human LTMC system to quantitate the frequency of CAFC (week 5) in aplastic anemia (AA). In bone marrow mononuclear cells (BM-MNC) of healthy donors (n = 36) we observed a mean frequency of 84.4 CAFC per 10(5) BM-MNC (95% confidence interval limits,66.4 to 102.4). The mean frequency of CAFC in BM of 31 AA patients was 6.6 per 10(5) BM-MNC (95% confidence interval limits,5.3 to 7.9; n = 47). This frequency is significantly lower as compared with controls (P textless .0001). The frequency of CAFC was reduced not only in pancytopenic AA patients (6.2 per 10(5) BM-MNC; P textless .0001 v control),but also in patients in remission after immunosuppression (7.6; P textless .0001 v control; P = .1 v pancytopenic AA patients). The CAFC frequency did not correlate with the severity or duration of the disease and did not predict response to immunosuppressive treatment. In summary,the frequency of primitive hematopoietic progenitor cells,as measured by the CAFC assay,is significantly reduced in AA. CAFC remain severely reduced even after hematologic recovery after immunosuppressive treatment. The low frequency of CAFC in remission patients is in keeping with other data pointing to a persisting defect of hematopoiesis in patients in remission after immunosuppressive treatment. View Publication

In a number of cell types,elevation of intracellular cAMP concentrations antagonizes growth factor-induced mitogenesis by abrogating the downstream signaling of RasGTP to extracellular-signal-regulated kinases (Erk 1,2). We studied the effect of elevation of cAMP concentrations on the IL-3-induced mitogenic response in the leukemic cell line AML193. We observed that 8-bromo-cAMP (8-Br-cAMP) had no inhibitory effect on the magnitude of this response. On the contrary. 8-Br-cAMP alone induced a proliferative response in these cells. 8-Br-cAMP activated Erk 1,2 in these cells without involvement of Shc phosphorylation. These findings suggest the presence of a novel cAMP-dependent signaling pathway in AML193 cells,which activates Erk 1,2 via a Shc-independent pathway and leads to the generation of a mitogenic response. View Publication

The Ly-49 family consists of at least nine members,of which Ly-49A and C have been found to be NK-cell inhibitory receptors specific for class I MHC. The functions of other Ly-49 molecules are still unclear. Further analysis of Ly-49 is complicated by the cross-reactivities of some anti-Ly-49 antibodies initially thought to be specific for individual Ly-49 molecules. Studies on the role of Ly-49 in hybrid resistance as well as on allelic exclusion are also complicated by our recent finding that a novel Ly-49CB6 gene is the likely allelic form of Ly-49CBALB as opposed to a previously reported highly related but distinct gene in B6 mice. In cell-cell binding assays,only Ly-49A and C show significant binding to class I MHC. Ly-49A and C also bind some polysaccharides,and carbohydrates on class I MHC seem to be important for its binding to Ly-49. However,this interaction involves not only the carbohydrate recognition domain of Ly-49 but also a part of the stalk region,suggesting that both carbohydrates and peptide backbone of class I MHC may be recognized by Ly-49. It is likely that additional Ly-49 molecules yet to be identified function as NK-inhibitory receptors specific for class I MHC. View Publication

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology,flow cytometry,and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR,G418 resistance,and X-gal staining was also within the expected range; 65%,44% and 23%,respectively. Thus,there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients,expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase,and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient,individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer. View Publication

Embryonic stem cells,derived from the inner cell mass of murine blastocysts,can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes,such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta,during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes. View Publication

Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-,atrium- and ventricle-like type,which can be distinguished by their specific patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes,we studied gene expression of beta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected,which revealed an increase of beta-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA,both,in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of beta-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/beta-galactosidase activity reflects an increase of cardiac- and ventricle-specific gene expression,a semi-quantitative RT-PCR analysis was performed for alpha-cardiac myosin heavy chain (alpha-MHC) and MLC-2v genes. It was shown that both 10(-8) M and 10(-9) M RA resulted in an increased level of alpha-cardiac MHC and MLC-2v mRNA in embryoid bodies in early,but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- and ventricle-like properties was shown to be increased by RA,whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected. View Publication

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays,knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells,respectively,as well as a 2- to 4-fold increase in SRC after 4 d of culture. However,after 9 d of culture,all SRC were lost,despite further increases in total cells,CFC content,and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation,and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells. View Publication