BackgroundMigration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs.Methodology/Principal FindingsA complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased.Conclusions/SignificanceUndifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.

Photoreceptor cell death occurs during both normal and pathological retinal development. We tested for selective induction and blockade of cell death in either retinal photoreceptors or their precursors. Organotypical retinal explants from rats at postnatal days 3-11 were treated in vitro for 24 hr with thapsigargin, okadaic acid, etoposide, anisomycin, or forskolin. Explant sections were examined for cell death, and identification of either photoreceptors or proliferating/immediate postmitotic cells followed imunohistochemistry for either rhodopsin or bromodeoxyuridine and proliferating cell nuclear antigen, respectively. Photoreceptor cell death was selectively induced by either thapsigargin or okadaic acid, whereas death of proliferating/immediate postmitotic cells was induced by etoposide. Prelabeling of proliferating precursors allowed direct demonstration of changing sensitivity of photoreceptors to various chemicals. Degeneration of both photoreceptors and proliferating/immediate postmitotic cells depended on protein synthesis. Increase of intracellular cyclic AMP blocked degeneration of postmitotic, but not of proliferating, photoreceptor precursors. The selective induction and blockade of cell death show that developing photoreceptors undergo progressive changes in mechanisms of programmed cell death associated with phenotypic differentiation.

1. We investigated the association of c-Jun with apoptosis within retinal tissue. Explants of the retina of neonatal rats were subject to a variety of procedures that cause apoptosis of specific classes of retinal cells at distinct stages of differentiation. The expression of c-Jun was detected by Western Blot, and immunohistochemistry was done with antibodies made for either N-terminal or C-terminal domains of c-Jun, and correlated with apoptosis detected either by chromatin condensation or by in situ nick end labeling of fragmented DNA. 2. c-Jun protein content was increased in retinal tissue subject to induction of both photoreceptor and ganglion cell death. 3. c-Jun N-terminal immunoreactivity was found mainly in the cytoplasm of apoptotic cells regardless of cell type, of the stage of differentiation, including proliferating cells, or of the means of induction of apoptosis. 4. The data are consistent with the hypothesis that c-Jun is involved in the control of cell death in retinal tissue, but other proteins that cross-react with c-Jun N-terminal antibodies may also be major markers of retinal apoptosis. 5. Antibodies directed to c-Jun N-terminal (aa 91-105) are useful tools to follow apoptotic changes in retinal tissue.

Astroglial cells are involved in directional movements of neurons such as migration of the neuronal cell body and growth of neurites. In the mammalian midbrain, medial (M) and lateral (L) radial glia and derived astrocytes differ in their ability to support neuritic growth. In previous work, we have demonstrated that the growthpermissive ability of L astrocytes and non-permissive properties of M astrocytes correlate with the respective composition of the cell surface-associated and secreted glycosaminoglycans (GAGs). Recent work also shows that the GAG-degrading enzyme heparitinase I increases the neurite growth-promoting ability of M midbrain astrocytes (Garcia-Abreu J et al. 2000 Glia 29: 260). In agreement with previous AFM studies of living glial cell lines and cells in primary culture, imaging of living L and M cells at similar load forces showed structures identified as F-actin fibers. Moreover, no systematic differences were observed between L and M pictures. By contrast, the surfaces of formaldehyde-fixed lateral (L) and medial (M) astrocytes differ by the presence of conspicuous 250 nm protrusions in the former, and of a fibrillar network in the extracellular matrix of the latter. Furthermore, we show that treatment with heparitinase I leads to disappearance of the fibrils from M cells and, consequently, to the assumption of an L-like appearance. Our results suggest that the formation of fibrils may follow from an ability to form large aggregates by association of HS carbohydrate units as has been unexpectedly detected by AFM for oligomers of polysialic acid or polysialic acid-containing carbohydrate units of N-CAM, with formation of filament bundles (Toikka J et al. 1998. J Biol Chem 273: 28557). Taken together with the functional effects of heparitinase I treatment, our present results demonstrate an important role of the extracellular matrix on the functional properties of astrocytes. They also emphasize the power and potentialities of AFM in the study of the extracellular matrix on the surface of fixed cells. - ( June 27, 2000 ) . * Supported by PRONEX/ MCT, CNPq, FAPERJ, CEPG/UFRJ. E-mail: gweissmuller@hotmail.com Aggregation of amyloid β peptide (Aβ) into fibrils and deposition as senile plaques are primarily related to neurotoxicity in Alzheimer's Disease (AD). Thus, agents interfering with aggregation may be potentially useful in preventing or decreasing Aβ toxicity. We first studied the effects of guanidine hydrochloride and temperature on the stability of fibrillar Aβ using peptides truncated at different positions. These experiments suggested that hydrophobic interactions mediated by the C-terminal portion of Aβ are important for fibril stability. Based on these results, we found two compounds capable of disaggregating amyloid fibrils at micromolar concentrations, as indicated by light scattering measurements and transmission electron microscopy. When applied to primary cultures of E18 rat hippocampal neurons, both drugs significantly reduced Aβ-induced cell death (as assayed by trypan blue ...