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At least 307 records · Page 17Linked to original sources
PubMed2001

A model of human p210(bcr/ABL)-mediated chronic myelogenous leukemia by transduction of primary normal human CD34(+) cells with a BCR/ABL-containing retroviral vector.

Most insights into the molecular mechanisms underlying transformation by the p210(BCR/ABL) oncoprotein are derived from studies in which BCR/ABL cDNA was introduced into hematopoietic or fibroblast cell lines. However, such cell line models may not represent all the features of chronic myelogenous leukemia (CML) caused by additional genetic abnormalities and differences in the biology of cell lines compared with primary hematopoietic progenitor and stem cells. A primary human hematopoietic progenitor cell model for CML was developed by the transduction of b3a2 BCR/ABL cDNA in normal CD34(+) cells. Adhesion of BCR/ABL-transduced CD34(+) cells to fibronectin was decreased, but migration over fibronectin was enhanced compared with that of mock-transduced CD34(+) cells. Adhesion to fibronectin did not decrease the proliferation of BCR/ABL-transduced CD34(+) cells but decreased the proliferation of mock-transduced CD34(+) cells. This was associated with elevated levels of p27(Kip) in p210(BCR/ABL)-expressing CD34(+) cells. In addition, the presence of p210(BCR/ABL) delayed apoptosis after the withdrawal of cytokines and serum. Finally, significantly more and larger myeloid colony-forming units grew from BCR/ABL than from mock-transduced CD34(+) cells. Thus, the transduction of CD34(+) cells with the b3a2-BCR/ABL cDNA recreates most, if not all, phenotypic abnormalities seen in primary CML CD34(+) cells. This model should prove useful for the study of molecular mechanisms associated with the presence of p210(BCR/ABL) in CML.

Antigens, CD34↗
PubMed2003

Glial cells generate neurons--master control within CNS regions: developmental perspectives on neural stem cells.

A common problem in neural stem cell research is the poor generation of neuronal or oligodendroglial descendants. The author takes a developmental perspective to propose solutions to this problem. After a general overview of the recent progress in developmental neurobiology, she highlights the necessity of the sequential and hierarchical specification of CNS precursors toward the generation of specific cell types, for example, neurons. In the developing as well as the adult CNS, multipotent stem cells do not directly generate neurons but give rise to precursors that are specified and restricted toward the generation of neurons. Some molecular determinants of this fate restriction have been identified during recent years and reveal that progression via this fate-restricted state is a necessary step of neurogenesis. These discoveries also demonstrate that neuronal fate specification is inseparably linked at the molecular level to regionalization of the developing CNS. These fate determinants and their specific action in distinct region-specific contexts are essential to direct the progeny of stem cells more efficiently toward the generation of the desired cell types. Recent data are discussed that demonstrate the common identity of precursors and stem cells in the developing and adult nervous system as radial glia, astroglia, or non-myelinating glia. A novel lineage model is proposed that incorporates these new views and explains why the default pathway of stem cells is astroglia. These new insights into the cellular and molecular mechanisms of neurogenesis help to design novel approaches for reconstitutive therapy of neurodegenerative diseases.

Animals↗
PubMed2004

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells.

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.

Animals↗
PubMed2004

Tissue-engineered bone via seeding bone marrow stem cell derived osteoblasts into coral: a rat model.

In the present study, natural coral of porites species was used as scaffold combined with in vitro expanded bone marrow stem cell derived osteoblasts (BMSC-DO), to develop a tissue-engineered bone graft in a rat model. Coral was molded into the shape of rat mandible seeded with 5x10(6) /ml BMSC-DO subsequently implanted subcutaneously in the back of 5 week Sprague dawely rats for 3 months. Coral alone was implanted as a control. The implants were harvest and processed for gross inspection and histological observations. The results showed that newly bone grafts were successfully formed coral seeded with cells group showed smooth highly vascularized like bone tissue. Histological sections revealed mature bone formation and lots of blood vessel, the bone formation occurred in the manner resemble intramembraneous bone formation. This study demonstrates that coral can be use as a suitable scaffold material for delivering bone marrow mesenchymal stem cells in tissue engineering.

Animals↗
PubMed1992

Isolation of a stem cell for neurons and glia from the mammalian neural crest.

We have isolated mammalian neural crest cells using a monoclonal antibody to the low affinity NGF receptor, and established conditions for the serial propagation of these cells in clonal culture to assess their developmental potential. This analysis indicates that, first, single mammalian neural crest cells are multipotent, able to generate at least neurons and Schwann cells like their avian counterparts. Second, multipotent neural crest cells generate multipotent progeny, indicating that they are capable of self-renewal and therefore are stem cells. Third, multipotent neural crest cells also generate some clonal progeny that form only neurons or glia, suggesting the production of committed neuroblasts and glioblasts. Manipulation of the substrate alters the fate of the multipotent cells. These findings have implications for models of neural crest development in vivo, and establish a system for studying the generation of cellular diversity by a multipotent stem cell in vitro.

Animals↗
PubMed1998

A dynamic model of proliferation and differentiation in the intestinal crypt based on a hypothetical intraepithelial growth factor.

A widely accepted model of the temporal and spatial organization of proliferation and differentiation in intestinal epithelial is based on a cellular pedigree with all cells descending from a few active stem cells and undergoing a sequence of transitory divisions until the non-proliferating maturing cell stages develop. Model simulations have shown that such a pedigree concept can explain a large variety of data. However, so far there is neither a direct experimental proof for the existence of an intrinsic age structure in the transitory proliferative cell stages nor for the distinction between stem and transitory cells. It is our objective to suggest an alternative model which is based on evidence for intercellular communications such as might be mediated through gap junctions. We consider the diffusion of a hypothetical intraepithelial growth factor in a chain of cells which are connected via gap junctions. Individual cells can divide if a critical growth factor concentration is exceeded. Simulation studies show that the model is consistent with many observed features of the small intestinal crypt in steady state and after perturbation.

Animals↗
PubMed2001

[Embryonic stem cells and therapeutic cloning].

Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.

Adult↗
PubMed2001

Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice.

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 x 10(5) VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.

Animals↗
PubMed1993

In vivo development of an acetyldinaline resistant subline of the BN rat acute myelocytic leukemia (BNML).

The cytostatic drug acetyldinaline [ACD, CI-994, 4-acetylamine-N-(2-aminophenyl)-benzamide] shows an extreme antileukemic effect in the Brown Norway (BN) rate model for acute myelocytic leukemia (BNML) with only minor toxicity for normal pluripotent hemopoietic stem cells. So far, the mode of action is unknown. A resistant subline (BNML/ACD-R) was developed in vivo in the BNML model. Leukemic rats received repeated oral administrations of ACD. When the leukemia relapsed after initial remission-induction with ACD, the cells were transferred to new recipients which were again treated. In total, the animals received 247 oral administrations of ACD (33 x 2 mg/kg per day and 214 x 5 mg/kg per day) before full resistance was reached. The cell line was transferred 17 times in total. Treatment of the final resistant cell line with therapeutically highly active doses of 23.7 mg/kg per day and 11.85 mg/kg per day ACD for 5 days, that resulted in an increase of life span (ILS) of 57 and 18 days, respectively, when applied to the sensitive parent BNML line (BNML/S), resulted in only 10 and 3 days ILS, respectively. These results indicate that a significant degree of resistance has been achieved, which can be overcome partially by increasing the dose of ACD. Whether the development of a resistant subpopulation of the BNML is a result of acquired resistance or whether a naturally resistant subpopulation has been selected out after prolonged treatment with ACD remains to be established. The currently available resistant subline BNML/ACD-R now offers the possibility for further studies on the mechanism of action of ACD.

Animals↗
PubMed1997

Embryonic stem cell lines from MRL mice allow genetic modification in a murine model of autoimmune disease.

The MRL/MpJ-Fas(lpr) (MRL-lpr/lpr) mouse spontaneously develops a generalized autoimmune disease with features similar to those of systemic lupus erythematosus. This mouse strain provides a valuable system for identifying and characterizing the multiple genetic factors that influence the pathogenesis of autoimmune diseases. One of the most powerful means of examining the role of a specific gene product in vivo is by inactivating the gene in mouse embryonic stem (ES) cells by homologous recombination and using these cells to derive mouse lines carrying the inactivated gene. The successful application of this approach, however, requires an ES cell line that will remain stable in culture during the processes of genetic manipulation and selection. To date, ES cell lines that meet this criterion have been derived from only a few mouse strains. Here we describe the production and characterization of stable ES cell lines from the MRL mouse strain. Approximately 7% of the blastocysts derived from the MRL/MpJ+ (MRL-+/+) strain gave rise to ES cell lines, and both of the male MRL-+/+ ES cell lines tested were shown to be germline competent. We show that the MRL-+/+ ES cell lines undergo gene targeting by homologous recombination at high frequency by inactivating the gene encoding the EP2 prostaglandin receptor. These Ep2-targeted MRL ES cell lines were used to generate MRL mouse lines heterozygous for the disrupted Ep2 gene, thus demonstrating the feasibility of using a genetic approach to dissect the pathobiology of the autoimmune disease in the MRL mouse.

Animals↗
PubMed1998

I-SceI-induced gene replacement at a natural locus in embryonic stem cells.

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 x 10[-6]) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

Animals↗
PubMed1997

A two-stage model for childhood acute lymphoblastic leukemia: application to hereditary and nonhereditary leukemogenesis.

A differential equation model is developed to represent a two-stage mutational process leading to childhood acute lymphoblastic leukemia (ALL). Leukemogenesis is modeled as transformation of target stem cells that initially grow rapidly in the embryo but plateau and then decline in postnatal childhood. Inheritance of the first of two leukemogenic mutations is allowed as a possibility in a small minority of leukemic patients who would characteristically develop leukemia at an early age. The model is shown to be capable of providing good fits to incidence data for childhood ALL; these fits allow estimation of some parameters of the model. The analysis shows that individuals inheriting one of the two mutations necessary for ALL would be likely to experience "multiclonal leukemogenesis"; that is, the parallel development of several leukemic clones arising from multiple independent leukemic events. The model suggests that between two and ten such clones would typically have developed in such individuals by the time of diagnosis. The main conclusions of the deterministic investigation were confirmed by stochastic modeling. The existence of multiclonal leukemogenesis is in principle testable by molecular biological methods (clonality analysis) that rely on the random inactivation of one of two X-chromosomes in normal female subjects. It is expected that the mathematical methods developed here will also be useful for more general (N-stage) models of malignant transformation of stem cell populations undergoing growth or decline.

Aging↗
PubMed2002

The in vivo development of human T cells from CD34(+) cells in the murine thymic environment.

There is increasing evidence that human hematopoietic stem cells can develop into lymphocytes expressing T cell surface markers in the organ culture of murine embryonic thymic lobes. If human T cells with functional maturity are inducible from human stem cells in the mouse, it may be a useful model to investigate human T cell development and the human immune response in vivo. To approach this, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood-derived CD34(+) cells (hu/m cluster) using reaggregate thymic organ culture, and subsequently implanted it under the kidney capsule of NOD/SCID mice. The implanted hu/m cluster grew in volume under the kidney capsule and contained increased numbers of CD4(+)CD8(+)cells as well as CD4 or CD8 single-positive cells with low CD1a expression. These lymphocytes were also shown to possess activity for producing IL-2 and IL-4. Characteristics similar to human T cells also developed in the thymus of newly established mice lacking NK activity from NOD/SCID mice. These results indicate that functionally mature T cells can develop in vivo from human hematopoietic progenitors in the murine environment composed of thymic epithelial cells.

Animals↗
PubMed2004

Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway.

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.

Cell Differentiation↗
PubMed2004

Milestones of neuronal development in the adult hippocampus.

Adult hippocampal neurogenesis originates from precursor cells in the adult dentate gyrus and results in new granule cell neurons. We propose a model of the development that takes place between these two fixed points and identify several developmental milestones. From a presumably bipotent radial-glia-like stem cell (type-1 cell) with astrocytic properties, development progresses over at least two stages of amplifying lineage-determined progenitor cells (type-2 and type-3 cells) to early postmitotic and to mature neurons. The selection process, during which new neurons are recruited into function, and other regulatory influences differentially affect the different stages of development.

Adult↗
PubMed1997

Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies.

Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a manner which could severely compromise the interpretation of the mutant phenotype. The present overview summarizes the literature on a wide variety of behavioral traits for the 129, C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field activity, learning and memory tasks, aggression, sexual and parental behaviors, acoustic startle and prepulse inhibition, and the behavioral actions of ethanol, nicotine, cocaine, opiates, antipsychotics, and anxiolytics. Using the referenced information, molecular geneticists can choose optimal parental strains of mice, and perhaps develop new embryonic stem cell progenitors, for new knockouts and transgenics to investigate gene function, and to serve as animal models in the development of novel therapeutics for human genetic diseases.

Animals↗
PubMed2004

Hematopoiesis in the feline fetal liver: an assessment by flow cytometry.

The domestic cat is an excellent model for the development of therapeutic protocols that target hematopoietic stem cells (HSCs) because it is relatively resistant to complications related to bone marrow transplantation. To identify a plentiful source of HSC that could be used as targets for gene transduction and transplantation, the livers of 28 mid-gestation fetuses (28-52 days) and late-gestation fetuses (53 days-term) were analyzed for erythroid, myeloid, lymphoid, and uncommitted hematopoietic progenitor cells by flow cytometry. We found that the fetal liver mononuclear cells (FLMCs) contained 57% erythroid progenitors during mid-gestation, but this percentage declined to 43% as gestation progressed. Myelomonocytic cells within FLMC were more numerous in late-gestation (31%) than in mid-gestation (18%). Two monoclonal antibodies (mAb), CH 152 and CH 755, which recognize cells with the potential to reconstitute multilineage hematopoiesis in cats, were tested. Approximately, 32% of FLMC from late-gestation fetuses expressed the epitope recognized by mAb CH 152, a significant increase above the 12% positive cells in mid-gestation fetuses. Approximately, 33% of hepatic mononuclear cells expressed the epitope recognized by mAb CH 755 in both mid-term and late-term fetuses. When expressed in absolute numbers, medians of 2.7 x 10(7) CH 152-positive cells and 3.2 x 10(7) CH 755-positive cells were extracted from the late-term fetal livers of individual cats. T-lymphocytes were a minor component (<3%) of FLMC, despite their presence in the thymus and spleen. These data suggest that the late-term feline fetal liver is a suitable source of mutipotential hematopoietic cells that could be used for gene therapy protocols in the cat.

Animals↗
PubMed1998

In vivo growth of transplanted genetically altered intestinal stem cells.

PURPOSE: Intestinal stem cell transplantation is a potential method of delivering genes to the small intestine. The authors have previously demonstrated the survival of transfected intestinal stem cells implanted into the rat small intestine. This study examines the growth of genetically altered intestinal stem cells that were grown on a polycarbonate membrane and implanted into the rat small intestine. METHODS: The IEC6 rat intestinal cell line serves as a model for intestinal stem cells. A subclone of the IEC6 cells was developed that stably expresses the lac Z gene introduced by a retroviral vector. The transfected cells were seeded at 500,000 cells/cm2 on a polycarbonate membrane. In 15 male Sprague-Dawley rats, a 0.75-cm enterotomy was created on the antimesenteric side of the small intestine 5 cm distal to the ligament of Treitz. A 0.5-cm2 segment of the membrane was sutured over the enterotomy with the cells facing away from the intestinal lumen. A segment of the omentum was wrapped around the patched enterotomy and sutured to the serosa. Three and 7 days after implantation, the implanted cells were retrieved, then fixed and stained with an X-gal solution. The number of cells was determined from the intensity of the X-gal staining. In five of the rats, frozen sections of the enterotomy sites were examined after staining with X-gal. RESULTS: All 15 rats survived the implantation procedure. Initially, 0.32 +/- 0.11 million cells were implanted. At both 3 and 7 days, the enterotomy site still contained viable transfected cells. The number of viable cells increased substantially to 1.52 +/- 0.46 and 3.52 +/- 1.87 million cells at 3 and seven days, respectively (P < .05). CONCLUSIONS: The polycarbonate membrane served as a good vehicle to efficiently deliver genetically altered intestinal stem cells to the small intestine. The transplanted cells continued to grow and stably expressed the gene product. This ex vivo approach to gene therapy has considerable therapeutic potential.

Animals↗