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Neuron. 2000 Oct;28(1):69-80.
Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells.

Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, Temple S.

Department of Pharmacology and Neuroscience, Albany Medical College, New York 12208, USA.

Multipotent stem cells that generate both neurons and glia are widespread components of the early neuroepithelium. During CNS development, neurogenesis largely precedes gliogenesis: how is this timing achieved? Using clonal cell culture combined with long-term time-lapse video microscopy, we show that isolated stem cells from the embryonic mouse cerebral cortex exhibit a distinct order of cell-type production: neuroblasts first and glioblasts later. This is accompanied by changes in their capacity to make neurons versus glia and in their response to the mitogen EGF. Hence, multipotent stem cells alter their properties over time and undergo distinct phases of development that play a key role in scheduling production of diverse CNS cells.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11086984&dopt=Abstract

IUBMB Life. 2000 Jul;50(1):63-8.
The calpain small subunit gene is essential: its inactivation results in embryonic lethality.

Zimmerman UJ, Boring L, Pak JH, Mukerjee N, Wang KK.

Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6068, USA. unjiail.med.upenn.edu

Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild-type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild-type littermates. Genotyping of fetuses at different stages of development also revealed only wild-type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11087123&dopt=Abstract

Dev Biol. 2000 Dec 1;228(1):19-28.
Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein.

Pratt T, Sharp L, Nichols J, Price DJ, Mason JO.

Department of Biomedical Sciences, Centre for Developmental Biology, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom.

We have generated embryonic stem (ES) cells and transgenic mice carrying a tau-tagged green fluorescent protein (GFP) transgene under the control of a powerful promoter active in all cell types including those of the central nervous system. GFP requires no substrate and can be detected in fixed or living cells so is an attractive genetic marker. Tau-tagged GFP labels subcellular structures, including axons and the mitotic machinery, by binding the GFP to microtubules. This allows cell morphology to be visualized in exquisite detail. We test the application of cells derived from these mice in several types of cell-mixing experiments and demonstrate that the morphology of tau-GFP-expressing cells can be readily visualized after they have integrated into unlabeled host cells or tissues. We anticipate that these ES cells and transgenic mice will prove a novel and powerful tool for a wide variety of applications including the development of neural transplantation technologies in animal models and fundamental research into axon pathfinding mechanisms. A major advantage of the tau-GFP label is that it can be detected in living cells and labeled cells and their processes can be identified and subjected to a variety of manipulations such as electrophysiological cell recording. 2000 Academic Press.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11087623&dopt=Abstract

Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13625-30.
Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.

Gronthos S, Mankani M, Brahim J, Robey PG, Shi S.

Craniofacial and Skeletal Diseases Branch, National Institute on Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.

Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules, and did not form adipocytes, whereas BMSCs routinely calcified throughout the adherent cell layer with clusters of lipid-laden adipocytes. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSCs formed lamellar bone containing osteocytes and surface-lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11087820&dopt=Abstract

Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13807-11.
Gene correction in hematopoietic progenitor cells by homologous recombination.

Hatada S, Nikkuni K, Bentley SA, Kirby S, Smithies O.

Departments of Pathology and Laboratory Medicine, and Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7525, USA.

Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. Corrected colonies were identified by their survival in selective medium after electroporation of correcting DNA into unfractionated mouse bone marrow cells and were confirmed by reverse transcription-PCR and sequencing. The observed frequency (4.4 +/- 3.3 x 10(-5) per treated clonogenic cell) is the same as in embryonic stem cells (2.3 +/- 0.4 x 10(-5)) with the same DNA and mutation. These data suggest that gene targeting to correct mutant genes eventually will prove feasible in HSCs capable of long-term bone marrow reconstitution.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11087825&dopt=Abstract

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