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Results for “stem cell model of development”

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PubMed1999

A genetically myeloablated MPS VII model detects the expansion and curative properties of as few as 100 enriched murine stem cells.

Causes of transplantation failures are often difficult to assess due to our inability to monitor hematopoietic stem cell (HSC) homing, distribution, and amplification in situ. We have developed a mouse model that permits histochemical localization of 1000-fold enriched HSC and quantification of their long-term expanded progeny in situ. The mice are genetically myeloablated (c-kit receptor mutated, W41/W41) and are beta-glucuronidase null (GUSB ; gus(mps)/gus(mps)). The GUSB- mice with mucopolysaccharidosis type VII (MPS VII), like a large number of human patients with similar diseases, have systemic lysosomal storage disease that leads to premature death. Congenic GUSB+, Lineage(lo), Sca-1(hi), c-Kit(hi), Hoechst(lo) HSC, at doses of 30, 100, 250, and 425 cells, implanted and amplified in adult W41/W41, gus(mps)/gus(mps) recipients in a dose-dependent manner. At autopsy, primary recipients of 100 and 425 donor cells had histologically identifiable donor GUSB+ cells in multiple sites and showed both myeloid and lymphoid expansion in bone marrow. Donor cells were rare in the liver and spleen of 100-cell recipients, but lysosomal storage was significantly reduced. The life span was significantly extended in engrafted recipients of 250 (36.7 +/- 3.84 weeks,p = 0.0316) and 425 (40.7 +/-1.53 weeks,p = 0.0033) cells compared to untreated mice (26.4 +/- 1.53 weeks). Secondary hosts of marrow from the recipients of 425 cells demonstrated continued expansion of the GUSB+ cells. Results indicate the genetically myeloablated MPS VII mice can be used to trace and enumerate donor cells long-term and to follow early engraftment events in situ.

Animals↗

PubMed2025

From genes to geometry: Controlling embryo models by programming genomic activation.

Embryo-like models derived from stem cells have emerged as powerful tools to study early development. In this issue, Lodewijk et al.1 demonstrate that activating just two enhancers via CRISPR activation (CRISPRa) in mouse embryonic stem cells (ESCs) can drive self-organization into structured embryo-like models, offering a genome-driven approach in stem cell and developmental biology.

Animals↗

PubMed2006

Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation.

Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. Here we identify three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. We conclude that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal.

Animals↗

PubMed2003

Conservation of a Pumilio-Nanos complex from Drosophila germ plasm to human germ cells.

Germ cells are the cells which ultimately give rise to mature sperm and eggs. In model organisms such as flies and worms, several genes that are required for formation and maintenance of germ cells have been identified and their interactions are rapidly being delineated. By contrast, little is known of the genes required for development of human germ cells and it is not clear whether findings from model organisms will translate into knowledge of human germ cell development, especially given observations that reproductive pathways may evolve more rapidly than somatic pathways. The Pumilio and Nanos genes have been especially well-characterized in model organisms and encode proteins that interact and are required for development of germ stem cells in one or both sexes. Here we report the first characterization of a mammalian Nanos homolog, human NANOS1 ( NOS1). We show that human NOS1 protein interacts with the human PUMILIO-2 (PUM2) protein via highly conserved domains to form a stable complex. We also show that in men, the NOS1 and PUM2 proteins are particularly abundant in germline stem cells. These observations mirror those in distant species and document for the first time a conserved protein-protein interaction in germ cells from flies to humans. These results suggest the possibility that the interaction of PUM2 and NOS1 may play a conserved role in germ cell development and maintenance in humans as in model organisms.

Animals↗

PubMed2006

Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses.

Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.

Animals↗

PubMedDate not supplied

Molecular signals regulating proliferation of stem and progenitor cells in mouse olfactory epithelium.

To understand how signaling molecules regulate the generation of neurons from proliferating stem cells and neuronal progenitors in the developing and regenerating nervous system, we have studied neurogenesis in a model neurogenic epithelium, the olfactory epithelium (OE) of the mouse. Our studies have employed a candidate approach to test signaling molecules of potential importance in regulating neurogenesis and have utilized methods that include tissue culture, in situ hybridization and mouse genetics. Using these approaches, we have identified three distinct stages of stem and transit amplifying progenitor cells in the differentiation pathway of olfactory receptor neurons (ORNs) and have identified mechanisms by which the development of each of these progenitor cell types is regulated by signals produced both within the OE itself and by its underlying stroma. Our results indicate that regulation of olfactory neurogenesis is critically dependent on multiple signaling molecules from two different polypeptide growth factor superfamilies, the fibroblast growth factors and the transforming growth factor beta (TGF-beta) group. In addition, they indicate that these signaling molecules interact in at least two important ways: first, opposing signals converge on cells at specific developmental stages in the ORN pathway to regulate proliferation and differentiation; and second, these signaling molecules--particularly the TGF-betas and their antagonists--play key roles in feedback loops that regulate the size of progenitor cell pools and thereby neuron number, during development and regeneration.

Animals↗

PubMed2006

Development of a mock hemopoietic stem cell component suitable for the validation of cryopreservation procedures.

We developed a laboratory model of a unit of hemopoietic progenitor cells (mock-HPC unit) suitable for the validation of HPC cryopreservation procedures. The project was prompted by the practical and ethical difficulty of using real HPC units collected from healthy donors and patients for validation purposes. Mock-HPC units of different volumes ranging from about 120 to about 540mL were prepared by pooling a routinely discarded by product of our procedure to prepare platelet concentrates from buffy-coats, a standard procedure in most blood centers in Europe. Five ABO/Rh identical buffy coats each of 50mL volume, obtained from 450mL whole blood units by hard spin, were pooled with 300mL of a commercial platelet additive solution. After soft spin, the supernatant platelet concentrate pool was removed. The bottom fraction of this procedure, which contains RBC, WBC, HPC and platelets, constitutes a mock-HPC unit of about 120mL volume. Several bottom fractions may be pooled to obtain a mock-HPC unit of the desired volume. We used 20 mock-HPC units to validate an automatic procedure of HPC cryopreservation with a controlled rate freezer. In particular, we documented the standardization of critical points of the cooling profile, such as the correspondence of the crystallization phase with the theoretical freezing temperature of the product, the temperature peak rise above the theoretical freezing temperature of -5.7 degrees C and the automatic achievement in the chamber of a constant minimum temperature during supercooling (about -37 degrees C) with mean chamber loading volumes ranging from 231.8 to 1027.2mL.

Blood Preservation↗

PubMed2004

Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.

3T3-L1 Cells↗

PubMed2006

Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm.

Formation of mesoderm from the pluripotent epiblast depends upon canonical Wnt/beta-catenin signaling, although a precise molecular basis for this requirement has not been established. To develop a robust model of this developmental transition, we examined the role of Wnt signaling during the analogous stage of embryonic stem cell differentiation. We show that the kinetics of Wnt ligand expression and pathway activity in vitro mirror those found in vivo. Furthermore, inhibition of this endogenous Wnt signaling abrogates the functional competence of differentiating ES cells, reflected by their failure to generate Flk1(+) mesodermal precursors and subsequent mature mesodermal lineages. Microarray analysis at various times during early differentiation reveal that mesoderm- and endoderm-associated genes fail to be induced in the absence of Wnt signaling, indicating a lack of germ layer induction that normally occurs during gastrulation in vivo. The earliest genes displaying Wnt-dependent expression, however, were those expressed in vivo in the primitive streak. Using an inducible form of stabilized beta-catenin, we find that Wnt activity, although required, does not autonomously promote primitive streak-associated gene expression in vitro. Our results suggest that Wnt signaling functions in this model system to regulate the thresholds or stability of responses to other effector pathways and demonstrate that differentiating ES cells represent a useful model system for defining complex regulatory interactions underlying primary germ layer induction.

Animals↗

PubMed2001

Variable maturation velocity and parameter sensitivity in a model of haematopoiesis.

We analyse an age-structured model for haematopoiesis, describing the development of specialized cells in the blood from undifferentiated stem cells and including the controlling effects of hormones. Variation in the length of time for maturing of precursor cells in this model has a stabilizing influence. When the maturing process does not vary, then the age-structured model reduces to a delay differential equation. Depending on the death process considered, either a differential equation with two time delays or a differential equation with a state-dependent delay is obtained. Each of these is analysed in turn, for its linear stability. A sensitivity analysis of the parameters in this model shows which biochemical processes in the negative feedback most strongly affect the solutions.

Anemia, Hemolytic↗

PubMed2000

Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice.

BACKGROUND: Transplantation of human hematopoietic stem cells is the only true test of their long-term repopulation potential. Models are readily available to evaluate murine hematopoietic stem cells, but few exist that allow reliable quantification of human stem cells. The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model enables quantification of human hematopoietic stem cells, but the conditions that permit human engraftment in all animals have yet to be defined. The aims of the project were, therefore, to describe the variables that allow human engraftment in the NOD-SCID mouse model and the techniques that accurately quantify this engraftment. METHODS: NOD-SCID mice that had or had not received 250, 325, or 400 cGy irradiation received cord blood (CB) mononuclear or CD34+ cells i.v. or i.p. Mice were killed 6 weeks after transplantation, and the bone marrow, spleen, and thymus were harvested. Four-color flow cytometric analysis, semi-quantitative PCR, myeloid and erythroid progenitor, and stem cell assays were used to monitor human engraftment. RESULTS: A 250 or 325 cGy and i.v. injection of CB mononuclear or CD34+ cells is required to detect multilineage human engraftment in the bone marrow, spleen, or thymus of NOD-SCID mice. Four-color flow cytometric analysis and semi-quantitative PCR enable accurate detection of 0.1% human cells. Progenitor and stem cell assays provide functional information about the engrafted cells. CONCLUSIONS: Successful development of the NOD-SCID mouse model and techniques to assess human engraftment now allow it to be used reliably to analyze the effects of short-term cytokine exposure on the long-term repopulating capacity of CB stem cells.

Animals↗

PubMed1998

Stem cell features of benign and malignant prostate epithelial cells.

PURPOSE: We present a new hypothesis suggesting that the different malignant potential of benign prostatic hyperplasia (BPH) and high grade prostatic intraepithelial neoplasia may be explained by distinct alterations in stem cell-like properties. MATERIALS AND METHODS: We used our results and the recent literature to develop this hypothesis in the context of an updated prostate stem cell model. RESULTS: While high grade prostatic intraepithelial neoplasia is a likely precursor lesion to many prostatic adenocarcinomas, BPH rarely if ever progresses directly to carcinoma. Prostate epithelium contains basal and secretory compartments. Secretory cells appear to differentiate from basal cells. Thus, prostatic stem cells most likely reside in the basal compartment. In BPH there is a slight increase in epithelial proliferation, yet most replicating epithelial cells within BPH maintain their normal restriction to the basal compartment. In high grade prostatic intraepithelial neoplasia there is a marked increase in cell proliferation. In contrast to BPH, the majority of proliferating cells in high grade prostatic intraepithelial neoplasia reside in the secretory compartment. The biological significance of this topographic infidelity of proliferation in high grade prostatic intraepithelial neoplasia remains unclear but may relate mechanistically to down regulation of the cyclin dependent kinase inhibitor, p27kip1. Normal basal cells express GSTP1, an enzyme that inactivates reactive electrophiles and organic hydroperoxides, and that may protect cells from deoxyribonucleic acid damaging agents. In contrast, normal secretory cells and high grade prostatic intraepithelial neoplasia cells do not express this enzyme. CONCLUSIONS: We propose that topographic infidelity of proliferation produces a population of secretory cells replicating in the absence of key genome protective mechanisms, thus setting the stage for an accumulation of genomic alterations and instability in high grade prostatic intraepithelial neoplasia. This action occurs along with activation of telomerase, resulting in an immortal clone capable of developing into invasive carcinoma. The model predicts that genome protection remains intact in BPH, minimizing its malignant potential.

Animals↗

PubMed1990

Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells.

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.

Animals↗

PubMed2006

Formation of human prostate tissue from embryonic stem cells.

Rodent models and immortalized or genetically modified cell lines are frequently used-but have limited utility-for studying human prostate development and maturation. Using rodent mesenchyme to establish reciprocal mesenchymal-epithelial cell interactions with human embryonic stem cells (hESCs), we generated human prostate tissue expressing prostate-specific antigen (PSA) within 8-12 weeks. This human prostate model shows species-conserved signalling mechanisms that could extend to integumental, gastrointestinal and genital tissues.

Animals↗

PubMed2004

Wnt proteins promote neuronal differentiation in neural stem cell culture.

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from studies of mouse and chick models, but its action at the cellular level has been poorly understand. In this study, we examine the in vitro function of Wnt signaling in embryonic neural stem cells, dissociated from neurospheres derived from E11.5 mouse telencephalon. Conditioned media containing active Wnt-3a proteins are added to the neural stem cells and its effect on regeneration of neurospheres and differentiation into neuronal and glial cells was examined. Wnt-3a proteins inhibit regeneration of neurospheres, but promote differentiation into MAP2-positive neuronal cells. Wnt-3a proteins also increase the number of GFAP-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4. These results indicate that Wnt-3a signaling can inhibit the maintenance of neural stem cells, but rather promote the differentiation of neural stem cells into several cell lineages.

Animals↗

PubMed2005

Differential expression of novel potential regulators in hematopoietic stem cells.

The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell-cell and cell-matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment.

Animals↗

PubMed2002

Efficacy and toxicity of a virus-directed enzyme prodrug therapy purging method: preclinical assessment and application to bone marrow samples from neuroblastoma patients.

Autologous stem cell transplantation is used to rescue cancer patients from myelosuppression caused by high-dose chemotherapy. However, autologous grafts often contain tumor cells that can contribute directly to relapse. Current purging methods are useful when fewer than 1% tumor cells contaminate the bone marrow, and patients with tumor burdens of >1% are considered ineligible for chemotherapy that necessitates stem cell rescue. Using neuroblastoma (NB) as a model system, we developed a method that is effective even with tumor burdens of 10-25%. Mixtures of NB-1691 NB cells and CD34(+) hematopoietic cells purged by this method showed no evidence of viable tumor cells as assessed by clonogenic assays or reverse transcription-PCR for the NB cell markers tyrosine hydroxylase and N-MYC. The efficacy and lack of toxicity of the method were verified using in vivo mouse models. Severe combined immunodeficient mice that received purged cell preparations containing 10% NB-1691 cells survived without evidence of disease for the observation period (>1 year), whereas mice that received unpurged cells developed disseminated disease requiring euthanasia 73-96 days after injection of cells. No evidence of toxicity to the mice was detected by numerous laboratory values for bone marrow, liver, and kidney function, and no difference was seen in the ability of purged cell mixtures versus unmanipulated CD34(+) cells to reconstitute the marrow of non-obese diabetic severe combined immunodeficient mice. In a pilot study, marrow was obtained from eight patients who had >/=1% metastatic tumor burden. All eight samples were purged to the level of detection by reverse transcription-PCR (samples from seven patients) or clonogenic potential (sample from one patient), whichever assay was used. The described adenovirus/rabbit carboxylesterase/CPT-11 (irinotecan, 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) virus-directed enzyme prodrug method may be useful for patients whose tumor burdens exceed 1% at the time of stem cell harvest. Assessment of purging efficacy with additional samples from NB patients is ongoing.

Adenoviridae↗

PubMed1998

EGL-17(FGF) expression coordinates the attraction of the migrating sex myoblasts with vulval induction in C. elegans.

During the development of the egg-laying system in Caenorhabditis elegans hermaphrodites, central gonadal cells organize the alignment of the vulva with the sex myoblasts, the progenitors of the egg-laying muscles. A fibroblast growth factor [EGL-17(FGF)] and an FGF receptor [EGL-15(FGFR)] are involved in the gonadal signals that guide the migrations of the sex myoblasts. Here we show that EGL-17(FGF) can act as an instructive guidance cue to direct the sex myoblasts to their final destinations. We find that egl-17 reporter constructs are expressed in the primary vulval cell and that EGL-17(FGF) expression in this cell correlates with the precise positioning of the sex myoblasts. We postulate that EGL-17(FGF) helps to coordinate the development of a functional egg-laying system, linking vulval induction with proper sex myoblast migration.

Animals↗