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Peter J Donovan

Publications and source records attributed to Peter J Donovan.

15 recordsLinked to original sources
PubMed2006

Neurotrophins mediate human embryonic stem cell survival.

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.

Brain-Derived Neurotrophic Factor↗
PubMed2006

Embryonic germ cells: when germ cells become stem cells.

Embryonic germ cells (EGCs) are pluripotent stem cells derived from primordial germ cells (PGCs). PGCs are progenitors of adult gametes, which diverge from the somatic lineage between late embryonic to early fetal development. First derived in the mouse, EGCs have also been derived from human, chicken, and pig. As pluripotent stem cells, EGCs demonstrate long-term self-renewal via clonal expansion in an undifferentiated state, and differentiate in vitro to form embryoid bodies containing cells that represent all three germ layers as well as mixed cell populations of less differentiated progenitors and precursors. This is also demonstrated in vivo by their formation into experimentally induced teratocarcinomas following transplantation. Furthermore, mice, pig, and chicken EGCs have also been shown to contribute to experimentally produced chimeric animals, including germline transmission. Importantly, EGCs demonstrate normal and stable karyotypes as well as normal patterns of genomic imprinting, including X-inactivation. Transplantation studies have begun in a variety of models in hopes of defining their potential use to treat a wide variety of human conditions, including diabetes and urological and neurological disorders.

Animals↗
PubMed2006

Staining embryonic stem cells using monoclonal antibodies to stage-specific embryonic antigens.

Stage-specific embryonic antigens (SSEAs) are cell-surface molecules that exhibit lineage-restricted patterns of expression during development. These antigens provide useful markers for identifying embryonic stem cells and their differentiated derivatives. SSEA-1 provides a surface marker for mouse and human primordial germ cells and mouse embryonic stem cells. SSEA-3 and SSEA-4 provide surface markers for human embryonic stem cells. Here, we describe methods for staining embryonic stem cells in culture and suspension using specific primary antibodies and fluorescent dye- or enzyme-conjugated secondary antibodies. Methods for alkaline phosphatase staining and immunohistochemical localization of antigens in paraffin sections are also described. Monoclonal antibodies directed to SSEAs are valuable research tools that can be used to: (1) determine the percentage of stem cells in mixed cultures; (2) examine the spatial distribution of stem cells and differentiated derivatives in monolayer cultures and embryoid bodies; and (3) isolate populations of differentiated and undifferentiated cells by immunomagnetic or fluorescence-activated cell sorting for cellular and molecular analyses.

Alkaline Phosphatase↗
PubMed2006

Oncostatin m maintains the hematopoietic microenvironment and retains hematopoietic progenitors in the bone marrow.

Bone marrow (BM) functions as the primary hematopoietic tissue throughout adult life by providing a microenvironment for the proliferation, differentiation, and retention of hematopoietic stem cells and progenitors. We describe novel roles for oncostatin M (OSM) in the BM hematopoietic microenvironment. Hematopoietic progenitor activity in OSM-deficient mice was reduced in BM but elevated in the spleen and peripheral blood. The level of circulating granulocyte colony-stimulating factor (G-CSF) was increased, whereas that of stromal cell-derived factor 1 (SDF-1) was decreased in OSM-deficient mice. Moreover, the ability of OSM-deficient BM stromal cells to support hematopoiesis in vitro was significantly reduced. These results indicate that OSM plays a unique role in hematopoiesis by maintaining the proper microenvironment for BM hematopoiesis; it also retains hematopoietic progenitors in BM by regulating G-CSF and SDF-1 levels.

Animals↗
PubMed2005

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases.

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.

Animals↗
PubMed2004

Chipping away at 'stemness'.

Global gene-expression analyses of human embryonic stem cells confirm the involvement of some known genes in stem-cell function and identify some new candidate regulators of stem-cell growth. Support remains elusive, however, for the concept of 'stemness'--a pattern of expression of genes that is common to all stem cells.

Cell Differentiation↗
PubMed2004

Essential function of oncostatin m in nociceptive neurons of dorsal root ganglia.

Oncostatin M (OSM) is a member of the interleukin-6 family of cytokines, and we have reported previously that the murine OSM receptor beta subunit (OSMR) was expressed in some neurons in the adult trigeminal and dorsal root ganglia (DRGs) and in the perineonatal hypoglossal nucleus. In the present study, we investigated the development of OSMR-positive neurons of DRGs in OSM-deficient mice. In situ hybridization revealed that OSMR-positive neurons in DRGs began to appear at postnatal day 0 (P0) and reached the adult level at P14. In the DRGs of the OSM-deficient mice, vanilloid receptor 1 (VR1)- and P2X3-positive small-sized neurons were significantly decreased. In addition, OSMR-positive neurons decreased, resulting in the reduced number of VR1/P2X3/OSMR-triple positive neurons. OSM-deficient mice displayed significantly reduced noxious responses in models of acute thermal, mechanical, chemical, and visceral pain. Thus, OSM plays an essential role in the development of a subtype of nociceptive neurons in the DRGs.

Animals↗
PubMed2003

Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis.

We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase. The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.

Anaphase↗
PubMed2003

Turning germ cells into stem cells.

Primordial germ cells (PGCs), the embryonic precursors of the gametes of the adult animal, can give rise to two types of pluripotent stem cells. In vivo, PGCs can give rise to embryonal carcinoma cells, the pluripotent stem cells of testicular tumors. Cultured PGCs exposed to a specific cocktail of growth factors give rise to embryonic germ cells, pluripotent stem cells that can contribute to all the lineages of chimeric embryos including the germline. The conversion of PGCs into pluripotent stem cells is a remarkably similar process to nuclear reprogramming in which a somatic nucleus is reprogrammed in the egg cytoplasm. Understanding the genetics of embryonal carcinoma cell formation and the growth factor signaling pathways controlling embryonic germ cell derivation could tell us much about the molecular controls on developmental potency in mammals.

Animals↗
PubMed2003

Determinants of retroviral-mediated gene delivery to mouse spermatogonia.

Spermatogonia represent a new route to transgenesis in mice and potentially in some commercially important domesticated animals. In addition, these cells are also a potential target for viral integration in patients receiving somatic cell gene therapy. But the factors influencing retroviral transduction into spermatogonia are not well understood. Because retroviral transduction is affected in part by the proliferative status of the host cell, we developed an improved cell culture system in which spermatogonia survive and proliferate for several days. We used this system to test the ability of a variety of murine and avian retroviruses to infect spermatogonia. We investigated the factors influencing retroviral transduction of spermatogonia, including the proliferative status of the infected cell, the type of viral envelope, the type of retroviral long terminal repeat, and the method of viral delivery. Here we show that many of the widely used retroviral vector systems can be used to successfully transduce spermatogonia at high efficiency. Moreover, we show that retroviral delivery of MDM2, the major downregulator of p53, promotes spermatogonial survival in culture, suggesting that p53 plays a role in regulating spermatogonial apoptosis induced by growth factor deprivation. These results further demonstrate the usefulness of this novel system of targeting substances of interest to the testis. These data have important implications for improving animal transgenesis and for understanding the risks associated with somatic cell gene therapy.

Animals↗
PubMed2002

Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer.

Establishment of the mammalian germ line is a prerequisite for fertility of the adult animal but we know surprisingly little about the molecular mechanisms regulating germ-line development in mammals. Signaling from the c-Kit receptor tyrosine kinase is essential for primordial germ cell (PGC) growth both in vivo and in vitro. Many downstream effectors of the c-Kit signaling pathway have been identified in other cell types but how these molecules control PGC survival and proliferation are unknown. Determination of the c-Kit effectors acting in PGCs has been hampered by the lack of effective methods to easily manipulate gene expression in these cells. We overcame this problem by testing the efficacy of retroviral-mediated gene transfer for manipulating gene expression in mammalian germ cells. We found that PGCs can be successfully infected with a variety of types of retroviruses. We used this method to demonstrate an important role for the AKT kinase in regulating PGC growth. Such technology for manipulating gene expression in PGCs will allow many of the molecular mechanisms regulating germ cell growth, behavior, and differentiation to be comprehensively analyzed.

Animals↗
PubMed2002

Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation.

In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF). The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response. We generated Cdc25b-deficient (Cdc25b-/-) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b-/- mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b-/- mice were sterile, and Cdc25b-/- oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b-/- oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b-/- female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.

Animals↗