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Results for “stem cell model of development”

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PubMed2000

Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells.

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron-like cells by a series of well-defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20-fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c-series gangliosides was downregulated concomitantly with upregulation of the expression of a- and b-series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA-induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development.

Animals↗
PubMed1995

[Early ontogeny of the human hematopoietic system].

Immunohistochemistry was used to detect markers of the vascular, stromal and hematopoietic cell compartments in the human embryo and early fetus, from 3 to 15 weeks of gestation. CD34 expression was consistently observed at the surface of vascular endothelial cells from off earliest stages tested, at the single exception of embryonic liver blood vessels. Yolk sac hematopoiesis was very transient and limited to primitive erythropoiesis. Clusters of erythroblasts, monocytes and granulocytes appeared from 4 to 5 weeks of gestation in the liver rudiment. The early development of the bone marrow was marked by the rapid invasion, at 8 weeks, of long bone cartilaginous rudiments by CD68+ osteoclast precursors, CD34+ endothelial cells and by preosteoblasts, leading to the development of large vascular sinuses between ossifying trabeculae. Endogenous erythro- and granulopoiesis developed from week 11 in primary logettes always organized around an arteriole, in a loose stromal mesenchymal network established between the media of these arterioles and the sinusal endothelium. Round, hematopoietic CD34+ cells were seen occasionally in yolk sac blood vessels. In the liver they were rare and intermingled as single cells in the hepatocyte cords; strikingly, CD34+ hematopoietic cells could seldom be detected in the developing bone marrow. In contrast, compact clusters of non-endothelial, round CD34+ CD45+ hematopoietic cells were detected, during the 5th week of development, in close association with the ventral wall of the dorsal aorta. These cells exhibit phenotypic and functional characteristics of very primitive hematopoietic progenitors. This observation is in striking correlation with the evidence accumulated in animal models that stem cells for the late embryonic and adult hematopoietic systems develop inside the embryo per se, in the vicinity of the dorsal aorta. We thus suggest that these aorta-associated CD34+ cells, that exhibit an anatomic localization similar to that of the intraembryonic stem cells identified in the avian and murine embryo, are the real stem of human hematopoiesis.

Bone Marrow↗
PubMed2005

Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.

Aging↗
PubMed2026

A reproducible three-dimensional model of human brain tissue to investigate physiological and disease-associated microglia phenotypes.

Stem-cell-based in vitro models offer promising potential to elucidate human brain cell functions and interactions, but limitations in reproducibility, maturation and cell-type diversity persist. Especially, prolonged incorporation of mature microglia and studies of neuroinflammation have proven challenging. Here, we developed a human induced pluripotent stem cell-based three-dimensional cortical brain tissue model (3BTM) containing neurons, astrocytes and microglia with high reproducibility, maturity and viability. 3BTMs show morphological, functional and proteomic maturation of all cell types, leading to high similarity to their in vivo counterparts. Incorporated microglia survive for over 6 months and display mature morphology, functions and gene expression. Importantly, when engineered to model Alzheimer's disease pathology, 3BTMs recapitulate key disease hallmarks, including amyloid deposition, increased phospho-tau levels and neuroinflammation, with microglia shifting their transcriptional landscape to disease-relevant signatures. Treatment of Alzheimer's disease 3BTMs with anti-Aβ immunotherapy cleared deposits and largely reversed disease signatures in glia. Together, our microglia-containing model provides a platform for studying physiological and pathological states of human brain tissue.

Humans↗
PubMed1993

Hematopoietic growth factor receptor genes as markers of lineage commitment during in vitro development of hematopoietic cells.

We have used two in vitro models to identify genes whose expression may serve as markers of lineage commitment during the development of hematopoietic stem cells. One system involves the development in vitro of blastocyst-derived embryonic stem cells into embryoid bodies. The second involves culturing of day 3.5 blastocysts in vitro under conditions that support their development into yolk saclike cysts. In both cases, hematopoietic cells arise in a manner that closely mimics the normal process occurring in the yolk sac of the early mouse embryo. We have focused our analysis on the expression of mRNAs for 15 hematopoietic growth factor receptor genes and other genes expressed in a hematopoietic lineage-specific manner. Although some growth factor receptor genes are apparently expressed constitutively during in vitro development, there are several classes of genes that undergo a highly consistent pattern of induction in both model systems. Genes induced early include those encoding the shared beta subunits of the interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors; those induced at intermediate times include the c-fms, G-CSF receptor, and CD34 genes; and a gene induced late during in vitro development is the IL-7 receptor gene. The defined temporal order for the expression of these genes suggests that they may be useful as markers for multiple stages in the development of different hematopoietic cell lineages during embryogenesis.

Animals↗
PubMed1990

A biological and computational model of megakaryocyte development as a stochastic branching process.

The purpose of this paper is to describe a model of megakaryocytopoiesis as a branching process with stochastic processes regulating critical control points of differentiation along the stem cell megakaryocyte platelet axis. Progress of cells through these critical control points are regulated by transitional probabilities, which in turn are regulated by influences such as growth factors. The critical control points include transition of resting megakaryocytic stem cells (CFU-meg) into proliferating stem cells, the cessation of cytokinesis, and the cessation of DNA synthesis. A computerized computational method has been developed for directly fitting the stochastic branching model to colony growth data. The computational model has allowed transitional probabilities to be derived from colony size data. The model provides a unifying explanation for much of the heterogeneity of stages of maturation within populations of megakaryocytes and is fully compatible with historical data supporting the stochastic nature of hematopoietic stem cell regulation and with modern molecular concepts about control of the cell cycle.

Cell Differentiation↗
PubMed2006

Derivation of human embryonic stem cells from developing and arrested embryos.

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.

Embryo, Mammalian↗
PubMed1988

Stem cell renewal and differentiation in acute myeloblastic leukaemia.

The defining properties of stem cells are capacities for self-renewal and, after determination, a limited number of terminal divisions. The blast cells of acute myeloblastic leukaemia (AML) are maintained by stem cells with these two properties. Since renewal and differentiation can be assessed separately in cultures of AML blasts, these cancer cells provide a useful model for examining stem regulation; such studies have practical importance for future developments in the treatment of AML. This paper considers three aspects of blast cell biology. First, evidence is presented that self-renewal and differentiation are regulated by specific genes; further, the DNA encoding these genes has structural features that affect the chemosensitivity of self-renewal. This sensitivity varies from patient-to-patient and is an important attribute contributing to variation in treatment efficacy. Second, the effects of myelopoietic growth factors on blast stem cells are presented and discussed, as these bear on the regulation of the balance between renewal and differentiation. Finally, models of leukaemic haemopoiesis are considered in light of the experimental findings. The suggestion is advanced that leukaemia can be explained better by abnormalities of gene expression than by blocked differentiation.

Antineoplastic Agents↗
PubMed2005

The zebrafish: a new model of T-cell and thymic development.

T-cell and thymic development are processes that have been highly conserved throughout vertebrate evolution. Mammals, birds, reptiles and fish share common molecular signalling pathways that regulate the development of the adaptive immune system. This Review article focuses on defining the similarities and differences between zebrafish and mammalian T-cell immunobiology, and it highlights the advantages of using the zebrafish as a genetic model to uncover mutations that affect T-cell and thymic development. Finally, we summarize the use of the zebrafish as a new model for assessing stem-cell function and for drug discovery.

Animals↗
PubMed2007

Niche-to-niche migration of bone-marrow-derived cells.

During ontogenesis, haematopoietic stem cells (HSCs) relocate between extra-embryonic and embryonic compartments. Similarly, site-specific homing of HSCs is ongoing during adulthood. With the expanding knowledge of HSC physiology, a new paradigm emerges in which HSCs and haematopoietic progenitor cells (HPCs) migrate to defined microenvironments within the bone marrow (BM) and to 'activated' or 'inducible' niches elsewhere. Here, we summarize current understanding of HSC niche characteristics, and the physiological and pathological mechanisms that guide HSC homing both within the BM and to distant niches in the periphery, promoting new vessel growth in tumours and ischaemia. Recent observations suggest that features of the HSC niche might also be recapitulated in pre-metastatic sites. Clusters of BM-derived HPCs promote invasion of disseminating cancer cells. Clear clinical benefits can be foreseen by modulating HSCs and their microenvironments, in promoting tissue regeneration, and inhibiting tumourigenesis and cancer metastasis.

Animals↗
PubMed2006

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Animals↗
PubMed2003

Retinoid target gene activation during induced tumor cell differentiation: human embryonal carcinoma as a model.

Many agents that exhibit chemopreventive activity are able to mediate a differentiation response in premalignant and malignant tissues. One of the most widely studied classes of tumor differentiation agents is the retinoids. There is rapidly evolving evidence for beneficial retinoid actions in the prevention or treatment of clinical tumors. However, the use of retinoids in the clinic is limited by acquired resistance and toxicity, especially when administered chronically in preventive strategies. Although retinoids are known to regulate gene transcription by activating retinoid receptors, the identity of the target genes that mediate the beneficial effects of retinoids are largely unknown. Here we review a useful model of retinoid-induced tumor cell differentiation: human embryonal carcinoma. The pluripotent nature and ease of use make human embryonal carcinoma cells a valuable and practical complement to human embryonic stem cells as an in vitro model of early human development. In addition, retinoid treatment of human embryonal carcinoma is an important model of induced tumor cell differentiation because retinoids cause the reversal of the malignant phenotype coincident with terminal neuronal differentiation. We have used both de novo and candidate approaches with this system in an effort to uncover critical downstream targets of retinoid receptors during differentiation induction.

Carcinoma, Embryonal↗
PubMed2004

Molecular and diagnostic clinical virology in real time.

During the last decade, the application of both qualitative and quantitative nucleic acid detection techniques has had a major impact on diagnostics in clinical virology. Both signal and target amplification-based systems are currently used routinely in most if not all virology laboratories. However, commercial assays are only available for a very limited number of targets, and this has resulted in the development and introduction of assays developed in-house for most viral targets. With improved and automated nucleic acid sample isolation techniques, as well as real-time detection methods, a new generation of assays for most clinically important viruses is being developed. These technological improvements also make it possible to generate results with a very short turnaround time. As an example of a more individual-patient disease-management concept, we have introduced in our clinical setting the quantitative detection of Epstein-Barr virus (EBV) in T-cell-depleted allogeneic stem cell transplant patients. This has enabled us to develop models for pre-emptive anti-B-cell immunotherapy for EBV reactivation, and for reducing not only the incidence of EBV lymphoproliferative disease (EBV-LPD), but the virus-related mortality. It is now also feasible to introduce molecular testing for those viruses that can easily be detected using classical virological methods, such as culture techniques or antigen detection. Prospective studies are needed to evaluate the clinical importance of the additional positive samples detected. It should, however, be clear that a complete exchange of technology is unlikely to occur, and that complementary methods should stay operational, making possible the discovery of new viruses. Furthermore, the ability to characterise viruses more easily by sequencing opens new possibilities for epidemiological studies. There is also an urgent need, with regard to molecular diagnostic methods, for the introduction and use of standardised materials and participation in international quality control programmes. Finally, with the introduction of a universal internal control throughout the whole procedure, the accuracy of the results generated is warranted.

DNA, Viral↗
PubMed2001

Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.

The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.

Actinin↗
PubMed1998

Immunotherapeutic aspects of allogeneic peripheral progenitor cells.

In a newly developed murine model of allogeneic peripheral progenitor transplantation (PBPCT) we investigated the immunotherapeutic potential of allogeneic peripheral stem cells. The following topics were addressed by our experiments: (1) comparison of the graft-versus-leukemia effect exerted by allogeneic PBPCT compared to allogeneic BMT; (2) the influence of T-lymphocytes on GVL activity; (3) the possibility to enhance the GVL activity of allogeneic PBPCT grafts by ex vivo cytokine incubation. Balb/c mice received cells of the syngeneic B-lymphatic leukemia A20 2 days prior to TBI (7.5 Gy) and the respective graft. The recipients received allogeneic bone marrow grafts or allogeneic peripheral progenitor cells obtained after mobilization of the donors (DBA/2) with either G-CSF in a dose of 250 microg/kg/day for 5 days. In some experiments T lymphocytes were removed by immunomagnetic depletion with CD3-coated beads. An additional group received T cell-depleted and IL-2/IL12-activated PBPCT grafts. The antileukemic activity of an allogeneic PBPCT graft was significantly greater than the antileukemic activity of an allogeneic BMT graft of the same size. Relapse rates were 80% in syngeneic PBPCT, 60% after allogeneic BMT and 34% after allogeneic PBPCT. This rise in antileukemic activity is not accompanied by a rise in GVHD mortality. Depletion of T lymphocytes by CD3-coated beads resulted in a nearly complete loss of the GVL activity with a relapse rate of 75%. Incubation of the T-depleted graft with IL-2 and IL-12 to enhance NK-based GVL activity has only limited success after MHC-matched transplantation with a relapse rate of 55%. Allogeneic PBPC exert a pronounced antileukemic effect. After MHC-matched PBPCT, this GVL effect resides mostly on the T cells of the graft. Ex vivo activation of T cell-depleted grafts by IL-2 and IL-12 is accompanied by an only limited reduction of relapse rate. PBPC are a valuable modality for primary transplantation in situations with high risk of relapse and for the treatment of relapse after BMT.

Animals↗
PubMed1996

Expansion of mutant stem cell populations in the human colon.

In general, it is presumed that colonic epithelial stem cells are the principal cell type at risk of incurring the series of somatic mutations leading to carcinoma, since all other epithelial cell types are short-lived. Mutant stem cell clonal expansion increases the risk for subsequent mutations and is therefore a potentially important step in carcinogenesis. The stem cells reside in colonic crypts, simple tubular foldings of the epithelium, and thus counting crypts provides an indirect means to determine stem cell numbers. The normal crypt population is known to expand through a process of crypt replication and this is thought to result in a corresponding expansion of the epithelial stem cell population. A simple mathematical model of the population dynamics of normal and mutant crypts (crypts containing mutant stem cells) is developed and used to estimate a lower bound on the relative rate of expansion of the mutant stem cell population. The model predicts that if mutant and normal crypt populations expand at the same rate, and if the mutation rate is small relative to the rate of growth, then the fraction of clusters of mutant crypts composed of only a single mutant crypt should steadily decrease with age towards one-half. Aberrant crypts are easily recognizable lesions in human colon which have frequently been shown to contain cells with K-ras and occasionally APC gene mutations. Application of the model to recent counts of aberrant crypt cluster sizes indicate that the aberrant crypt population, and the contained mutant stem cell population, is expanding substantially faster than normal.

Cell Division↗
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↗