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Clonal analysis of hematopoietic stem-cell differentiation in vivo.

Previous work has shown that the 0.02-0.05% of adult mouse bone marrow cells that bear the cell surface phenotype Thy-1loLin-Sca-1+ are enriched 1000- to 2000-fold for hematopoietic stem-cell activity in a variety of assays. When 50-100 cells of this phenotype are injected into an irradiated animal, they can permanently repopulate the entire hematopoietic system. In the present study, limiting-dilution and single-cell experiments were used to address the question of how individual Thy-1loLin-Sca-1+ stem cells contribute to repopulation of the hematopoietic system following irradiation. We calculated that 1 of 13 Thy-1loLin-Sca-1+ cells formed a clone comprising greater than 1% of peripheral white blood cells 3-7 weeks after injection. The majority of these clones included both lymphoid and myeloid lineages. Approximately one-third of the clones continued to produce new blood cells for 9 weeks or more, but the remainder disappeared earlier, including many that were multilineage. Thus, while the majority of Thy-1loLin-Sca-1+ bone marrow cells whose progeny are detected in the in vivo repopulation assay are pluripotential, only a subset undergo long-term self-renewal in vivo. Repopulation appears to be oligoclonal when limiting numbers of Thy-1loLin-Sca-1+ cells are injected. However, the number of clones contributing to hematopoiesis increases in proportion to the number of Thy-1loLin-Sca-1+ cells injected, bringing into question the notion that steady-state hematopoiesis in normal individuals is oligoclonal.

Animals↗

Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage.

We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.

Alkaline Phosphatase↗

Neural stem cell differentiation is dependent upon endogenous caspase 3 activity.

Caspase proteases have become the focal point for the development and application of anti-apoptotic therapies in a variety of central nervous system diseases. However, this approach is based on the premise that caspase function is limited to invoking cell death signals. Here, we show that caspase-3 activity is elevated in nonapoptotic differentiating neuronal cell populations. Moreover, peptide inhibition of protease activity effectively inhibits the differentiation process in a cultured neurosphere model. These results implicate caspase-3 activation as a conserved feature of neuronal differentiation and suggest that targeted inhibition of this protease in neural cell populations may have unintended consequences.

Animals↗

Dodeca-CLE peptides as suppressors of plant stem cell differentiation.

In plants and animals, small peptide ligands that signal in cell-cell communication have been suggested to be a crucial component of development. A bioassay of single-cell transdifferentation demonstrates that a dodecapeptide with two hydroxyproline residues is the functional product of genes from the CLE family, which includes CLAVATA3 in Arabidopsis. The dodecapeptide suppresses xylem cell development at a concentration of 10(-11) M and promotes cell division. An application, corresponding to all 26 Arabidopsis CLE protein family members, of synthetic dodecapeptides reveals two counteracting signaling pathways involved in stem cell fate.

Amino Acid Sequence↗

Role of Rac-GTPase and reactive oxygen species in cardiac differentiation of stem cells.

In the life of a cell, there is a constant balance between generation of reactive oxygen species (ROS) and activity of antioxidant defense mechanisms. Besides the damaging effects of ROS on many biomolecules, ROS also play a significant role in signal transduction pathways of growth factors suggesting a role of oxidative species in cell differentiation. ROS have recently been involved in the process of cardiac differentiation of stem cells. Several molecular mechanisms, including ones mediated by the GTPase Rac that underlie the regulatory role of ROS in the process of stem cell differentiation toward a cardiac lineage, are reviewed.

Animals↗

Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation.

The ability to generate a wide spectrum of differentiated cell types from ES cells in culture offers a powerful approach for studying lineage induction and specification and a promising source of progenitors for cell replacement therapy. Although significant efforts are being made to optimize culture conditions for the generation of different cell populations from ES cells, the identification and efficient isolation of specific progenitors for many lineages within these cultures remains a major challenge. By specifically tracking hematopoietic and cardiac development, we demonstrate here that these two lineages arise from distinct mesoderm subpopulations that develop in sequential waves from pre-mesoderm cells. Access to these populations provides a unique approach to isolate and characterize the earliest progenitors of these lineages.

Animals↗

Expression of the electrophysiological system during murine embryonic stem cell cardiac differentiation.

BACKGROUND: Stem cell based replacement therapy is envisioned as a method to repair failing hearts suffering from cardiomyocyte loss. To prevent potentially lethal arrhythmias, the donor cellular electrophysiological make-up should match with the acceptor tissue. To engineer the desired electrophysiological phenotype, the underlying molecular regulation of ion channels and gap-junction proteins should be clarified first. METHODS: We established the expression of seven main cardiac ion channel a-subunits and four b-subunits using semiquantitive RT-PCR and two major cardiac gap-junction proteins by immunohistochemistry during the differentiation process of murine ES cells into cardiomyocytes. RESULTS: Ion channel mRNA expression profiles display sequential upregulation. Connexin-40 and -43 expression is low in early cardiomyocytes, increased expression rates were found in subsequent differentiation phases. CONCLUSION: Cardiac differentiation of mouse embryonic stem cells is characterized by a sequential upregulation of the main cardiac ion channels and connexins.

Action Potentials↗