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Results for “stem cell model of development”

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PubMed2002

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases.

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Alkaline Phosphatase↗

PubMed2006

Differences between human embryonic stem cell lines.

The promise of human embryonic stem cell (hESC) lines for treating injuries and degenerative diseases, for understanding early human development, for disease modelling and for drug discovery, has brought much excitement to scientific communities as well as to the public. Although all of the lines derived worldwide share the expression of characteristic pluripotency markers, many differences are emerging between lines that may be more associated with the wide range of culture conditions in current use than the inherent genetic variation of the embryos from which embryonic stem cells were derived. Thus, the validity of many comparisons between lines published thus far is difficult to interpret. This article reviews the evidence for differences between lines, focusing on studies of pluripotency marker molecules, transcriptional profiling, genetic stability and epigenetic stability, for which there is most evidence. Recognition and assessment of environmentally induced differences will be important to facilitate the development of culture systems that maximize stability in culture and provide lines with maximal potential for safety and success in the range of possible applications.

Cell Culture Techniques↗

PubMed2004

JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells.

The AP-1 transcription factor JunB is a transcriptional regulator of myelopoiesis. Inactivation of JunB in postnatal mice results in a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML). Here, we show that JunB regulates the numbers of hematopoietic stem cells (HSC). JunB overexpression decreases the frequency of long-term HSC (LT-HSC), while JunB inactivation specifically expands the numbers of LT-HSC and granulocyte/macrophage progenitors (GMP) resulting in chronic MPD. Further, we demonstrate that junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD and that only junB-deficient LT-HSC are capable of transplanting the MPD to recipient mice. These results demonstrate a stem cell-specific role for JunB in normal and leukemic hematopoiesis and provide experimental evidence that leukemic stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of MPD.

Animals↗

PubMed2006

[Role of pulmonary stem cells labeled with bromodeoxyuridine and telomerase reverse transcriptase in hyperoxic lung injury in neonatal rats].

OBJECTIVE: To investigate characteristics of pulmonary stem cells labeled with bromodeoxyuridine (Brdu) and telomerase reverse transcriptase (TERT) in lung tissue, as well as the effects of proliferation and differentiation of the stem cells on lung development and repair of pulmonary injury. METHODS: A model of hyperoxia in neonatal rats was made by exposing the rats to 95% O2 for 7 d. Before sacrificing the rats, Brdu was injected through peritoneum, and immune staining positive cells were analyzed after the rats were sacrificed. TERT positive cells were stained by an immunohistochemical method. At the same time, the double staining for surfactant protein C (SPC) and Brdu or SPC and TERT were performed. Lung histologic study was done on HE stained tissue slices. RESULTS: (1) The lung with hyperoxic injury had thinner walls of alveoli, simple alveolar structure, fewer and larger alveoli, expanded and shrunken alveoli, and there were many fell-off alveolar epithelial cells in the alveolar cavities as well. (2) The cells positively stained with Brdu located in septa, mucosa and submucosa of various bronchi, scattering in epithelium of bronchi, and the number of positive cells was low, having a large nucleus. The TERT-positive cells were apparent in the septa and alveolar walls of peripheral lung tissue, characterized by uneven distribution in the lung lobes, the number of positive cells was less than that of Brdu-positive cells [integral of expression (1.61 +/- 0.83) vs. (0.62 +/- 0.55), P < 0.05]. The number of Brdu- and TERT-positive cells had no significant difference in hyperoxic rats compared to that in controls [integral of expression (1.43 +/- 0.85) vs. (1.61 +/- 0.83); (0.62 +/- 0.55) vs. (0.83 +/- 0.84), P > 0.05]. (3) After double staining, a few positive cells were found in double-stained tissues with SPC and Brdu or TERT. (4) The cells positively stained with SPC antibody had different size. The percentage of positive cells was not significantly different between the hyperoxia group (80.3%) and control group (78.6%). The Brdu positive staining located in nucleus of cells that had larger size than the cells not stained, round nucleus with intense staining (seldom, pole-shaped) and the number of such cells was less than that of the SPC positive cells. The percentage of positive cells was not significantly different between the hyperoxia group (28.5%) and control group (21.4%). (5) The TERT staining located in nucleus of cell that had smaller size than the cells not stained, various nuclear shape, including round intensively stained, round slightly stained, pole-shaped and divided shape. The percentage of positive cells was not significantly different between the hyperoxia group (2.3%) and control group (1.5%). CONCLUSIONS: (1) Brdu and TERT, as markers of stem cells having different capability of differentiation, possess special characteristics, respectively. The cells with Brdu could be transit amplifying cell (TAC) which retains characteristics of stem cells originated from differentiated stem cells, while, the cells stained with TERT especially reflects the characteristics of stem cells. (2) The proliferation and differentiation of pulmonary stem cells during hypoxic lung injury are limited and may be related with arrest of alveolization.

Alveolar Epithelial Cells↗

PubMed2006

Cardiovascular genomics: a current overview of in vivo and in vitro studies.

The cardiovascular system is the first system that is developed in the embryo. The cardiovascular development is a complex process involving the coordination, differentiation, and interaction of distinct cell lineages to form the heart and the diverse array of arteries, veins, and capillaries required to supply oxygen and nutrients to all tissues. Embryonic stem cells have been proposed as an interesting model system to investigate molecular and cellular mechanisms involved in mammalian development. The present review is focused on extrinsic soluble factors, intrinsic transcription factors, receptors, signal transduction pathways, and genes regulating the development of cardiovascular system in vivo and in vitro. Special emphasis has been given to cardiovascular genomics including gene expression studies on the cardiovascular system under developmental and pathophysiological conditions.

Animals↗

PubMed1999

Estimation of the target stem-cell population size in chronic myeloid leukemogenesis.

Estimation of the number of hematopoietic stem cells capable of causing chronic myeloid leukemia (CML) is relevant to the development of biologically based risk models of radiation-induced CML. Through a comparison of the age structure of CML incidence data from the Surveillance, Epidemiology, and End Results (SEER) Program and the age structure of chromosomal translocations found in healthy subjects, the number of CML target stem cells is estimated for individuals above 20 years of age. The estimation involves three steps. First, CML incidence among adults is fit to an exponentially increasing function of age. Next, assuming a relatively short waiting time distribution between BCR-ABL induction and the appearance of CML, an exponential age function with rate constants fixed to the values found for CML is fitted to the translocation data. Finally, assuming that translocations are equally likely to occur between any two points in the genome, the parameter estimates found in the first two steps are used to estimate the number of target stem cells for CML. The population-averaged estimates of this number are found to be 1.86x10(8) for men and 1.21x10(8) for women; the 95% confidence intervals of these estimates are (1.34x10(8), 2. 50x10(8)) and (0.84x10(8), 1.83x10(8)), respectively.

Adolescent↗

PubMed2003

Essential role for puma in development of postembryonic neural crest-derived cell lineages in zebrafish.

Multipotent neural crest stem cells have been identified in late gestation amniote embryos. Yet, significant questions remain about the mechanisms by which these cells are generated, maintained, and recruited during postembryonic development. The zebrafish, Danio rerio, offers an opportunity to identify genes essential for these processes, by screening for mutants with defects in traits likely to depend on these cells during metamorphosis and adult life. One such trait is the pigment pattern formed by neural crest-derived pigment cells, or chromatophores, which include black melanophores, yellow xanthophores, and iridescent iridophores. Previous analyses have demonstrated that the adult zebrafish pigment pattern depends on the de novo differentiation of latent precursor cells during both early and late phases of pigment pattern metamorphosis. To better understand the development of these cells, in this study, we analyze the zebrafish puma mutant, which ablates most of the adult melanophores that differentiate during metamorphosis, but leaves intact early larval melanophores that differentiate during embryogenesis. We use epistasis analyses to show that puma promotes the development of both early-appearing metamorphic melanophores that depend on the kit receptor tyrosine kinase, as well as late-appearing metamorphic melanophores that depend on both the G-protein-coupled endothelin receptor b1 (ednrb1) and the kit-related fms receptor tyrosine kinase. We further demonstrate that, during pigment pattern metamorphosis, puma mutants have deficiencies in the numbers of cells expressing transcripts for kit, ednrb1, and fms, as well as the HMG domain transcription factor sox10. Because the puma mutant phenotype is temperature-sensitive, we use temperature-shift experiments to identify a critical period for puma activity during pigment pattern metamorphosis. Finally, we use cell transplantations to show that puma acts cell-autonomously to promote the expansion of pigment cell lineages during metamorphosis. These results suggest a model for the lineage diversification of neural crest stem cells during zebrafish postembryonic development.

Animals↗

PubMed2005

Stem cells and brain cancer.

One of the most devastating CNS pathologies is brain cancer. The undifferentiated character of brain tumor cells and recent reports of cancer stem cells prompt questions regarding the involvement of normal stem/progenitor cells in brain tumor biology, their potential contribution to the tumor itself, and whether they are the cause or the consequence of tumor initiation and progression. The cancer stem cell model proposes a clonally derived brain tumor arising from a cancer stem cell. This tumor cell-of-origin originates from a stem/progenitor or more differentiated cell via acquisition of oncogenic mutations that dysregulate or allow reacquisition of self-renewal mechanisms. The tumor cells differentiate unidirectionally from the cancer stem cell in a way parallel to normal development. However, several properties of brain tumors add complexity to this model. For example, the apparent lineage and differentiation status of tumor cells are significantly affected by signaling abnormalities that are causally related to formation of the tumor. In addition, these tumors recruit normal CNS stem and progenitor cells to the tumor mass leading to the possibility of a heterogeneous and polyclonal cell population. It is likely that a complete description of the role of stem cells in brain tumors will be more complex than our current models.

Animals↗

PubMed2006

Human embryonic stem cells as a powerful tool for studying human embryogenesis.

Human embryonic stem cells (HESC) are pluripotent stem cell lines derived from the inner cell mass (ICM) of human blastocyst-stage embryos. They are characterized by their unlimited capacity to self-renew in culture. In addition, they have a broad developmental potential, as demonstrated by their ability to form practically any cell type in vivo and in vitro. These two features have made HESC extremely important in basic and applied research. In addition, they may serve as a powerful tool for studying human development. HESC can recapitulate embryogenesis by expressing developmentally regulated genes and by activating molecular pathways as they occur in vivo. Moreover, they can be used to analyze the effect of specific mutations on particular developmental events and may enable us to identify critical factors that play a role in the processes of cell commitment, differentiation, and adult cell reprogramming. Thus, modeling human embryogenesis by the use of HESC may allow new insights into developmental processes, which would otherwise be inaccessible for research.

Cell Differentiation↗

PubMed2001

Cloned transgenic mouse fetuses from embryonic stem cells.

Development of efficient efficient system for genetic modification and large-scale cloning of livestock is of importance for agriculture, biotechnology, or human medicine. The mouse, on the other hand, is an ideal model in the basic studies of genetic modification. In this study, we investigated about production of clone mice from established embryonic stem (ES) cell line by nuclear transfer. Further, we had try of production of cloned transgenic mouse fetuses/offspring using ES cells modified with a marker gene, EGFP. With the ES cell line TT2 which is at least 15 passages, reconstructed oocytes developed to 2-8 cell embryos, morulae, or blastocysts (44.8%), and 17.2% of them developed to term (19.5 days post-coitum, dpc). When 40 embryos with the marker gene transferred to 11 surrogate mothers (pseudopregnant females), 5 live fetuses were recognized in the uteli at 13.5 dpc and in these fetuses expression of GFP was observed, but none developed beyond 19.5 dpc. The present results suggest that ES cells can be used tg produce cloned mice.

Animals↗

PubMed2006

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract.

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.

Animals↗

PubMed2004

Fetomaternal cell traffic, pregnancy-associated progenitor cells, and autoimmune disease.

Fetal cells in maternal blood are a potential source of fetal genetic material that can be obtained non-invasively. Efforts to isolate these cells from maternal peripheral blood are limited by their low circulating numbers (approximately 1 per ml of maternal blood in euploid pregnancies). Expansion of these cells by culture would provide more cells for diagnosis and give an opportunity to study fetal metaphase chromosomes. Despite extensive optimization of culture conditions, many groups have failed reproducibly to grow fetal cells from pre-procedural maternal samples. An unexpected benefit of this research has been the discovery of a novel population of fetal cells, the pregnancy-associated progenitor cell (PAPC), which remains in maternal blood and tissue for decades following delivery. These cells might play a role in some autoimmune diseases, such as scleroderma. PAPCs appear to have stem cell characteristics, such as the ability to proliferate and differentiate. Recently developed animal models will help to ascertain whether these cells cause disease, respond to disease, or have therapeutic applications.

Adult↗

PubMed2000

Pax-5 and EBF are expressed in committed B-cell progenitors prior to the colonization of the embryonic bursa of fabricius.

The committed B-cell precursors developing from hemopoietic stem cells have been considered to differentiate through a common lymphoid progenitor stage in the mouse. In the chicken B-cell system, however, the committed B-cell progenitors burst as a single wave prior to the bursal colonization and most likely as direct descendants of hemopoietic stem cells. In the present report we show that prebursally committed B-cell progenitors specifically express early B-cell factor (EBF) and Pax-5 transcription factors. In addition we show that the expression of these and other B-lineage-associated transcription factors starts early in the chicken ontogeny. Altogether our findings strongly support the model of early delineation of B- and T-cell development and do not support the existence of common lymphoid stem cells.

Animals↗

PubMed2000

Complex organization in multicellularity as a necessity in evolution.

By introducing a dynamical system model of a multicellular system, it is shown that an organism with a variety of differentiated cell types and a complex pattern emerges through cell-cell interactions even without postulating any elaborate control mechanism. Such an organism is found to maintain a larger growth speed as an ensemble, by achieving a cooperative use of resources, than do simple homogeneous cells, which behave "selfishly." This suggests that the emergence of multicellular organisms with complex organization is a necessity in evolution. According to our theoretical model, there initially appear multipotent stem cells, which undergo stochastic differentiation to other cell types. With development and differentiation, both the chemical diversity and the complexity of intra-cellular dynamics are decreased, as a general consequence of our system. Robustness of the developmental process is also confirmed.

Biological Evolution↗

PubMed1999

Life without huntingtin: normal differentiation into functional neurons.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Animals↗

PubMedDate not supplied

Role of prosaposin in the male reproductive system: effect of prosaposin inactivation on the testis, epididymis, prostate, and seminal vesicles.

SGP-1/prosaposin can be secreted or targeted to the lysosomes where it is processed into smaller saposins (A, B, C, and D) required for the hydrolysis of glycosphingolipids. The deficiency of saposins B and C results in variant forms of metachromatic leukodystrophy and Gaucher's disease, respectively, which are characterized by lysosomal storage of undegraded glycosphingolipids. In the nervous system, prosaposin presents trophic activity. A mouse model was recently developed by creating a null allele in embryonic stem cells through gene targeting to investigate the phenotypic diversity of prosaposin mutations and the involvement of this protein in lysosomal storage diseases, and for the development of therapeutic approaches. Mice homozygous mutants die at the age of 35-40 days and neurological disorders contribute to the early demise of the mutant mice. The male reproductive organs in homozygous mutants show several abnormalities, such as a decrease in testis size with reduced spermiogenesis and an involution of the prostate, seminal vesicles, and epididymis. In these animals, the blood levels of testosterone remain normal. In the prostate of homozygous mutants, only the basal epithelial cells appear to be present, while the secretory cells are absent. These findings suggest that prosaposin may be involved in the development and maintenance of the male reproductive organs, as well as, in cellular differentiation.

Animals↗

PubMed2002

In vivo DNA electrotransfer.

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subcloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.

Animals↗

PubMed2005

Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling.

In the adult, the heart rate is driven by spontaneous and repetitive depolarizations of pacemaker cells to generate a firing of action potentials propagating along the conduction system and spreading into the ventricles. In the early embryo before E9.5, the pacemaker ionic channel responsible for the spontaneous depolarization of cells is not yet functional. Thus the mechanisms that initiate early heart rhythm during cardiogenesis are puzzling. In the absence of a functional pacemaker ionic channel, the oscillatory nature of inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+ signaling could provide an alternative pacemaking mechanism. To test this hypothesis, we have engineered pacemaker cells from embryonic stem (ES) cells, a model that faithfully recapitulates early stages of heart development. We show that InsP3-dependent shuttle of free Ca2+ in and out of the endoplasmic reticulum is essential for a proper generation of pacemaker activity during early cardiogenesis and fetal life.

Animals↗