Showing papers on "Neurosphere published in 1991"

Isolation and characterization of mouse neural precursor cells in primary culture.

Abstract: Primary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-D-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system.

Inductive differentiation of two neural lineages reconstituted in a microculture system from Xenopus early gastrula cells.

Abstract: Neural induction of ectoderm cells has been reconstituted and examined in a microculture system derived from dissociated early gastrula cells of Xenopus laevis. We have used monoclonal antibodies as specific markers to monitor cellular differentiation from three distinct ectoderm lineages in culture (N1 for CNS neurons from neural tube, Me1 for melanophores from neural crest and E3 for skin epidermal cells from epidermal lineages). CNS neurons and melanophores differentiate when deep layer cells of the ventral ectoderm (VE, prospective epidermis region; 150 cells/culture) and an appropriate region of the marginal zone (MZ, prospective mesoderm region; 5–150 cells/culture) are co-cultured, but not in cultures of either cell type on their own; VE cells cultured alone yield epidermal cells as we have previously reported. The extent of inductive neural differentiation in the co-culture system strongly depends on the origin and number of MZ cells initially added to culture wells. The potency to induce CNS neurons is highest for dorsal MZ cells and sharply decreases as more ventrally located cells are used. The same dorsoventral distribution of potency is seen in the ability of MZ cells to inhibit epidermal differentiation. In contrast, the ability of MZ cells to induce melanophores shows the reverse polarity, ventral to dorsal. These data indicate that separate developmental mechanisms are used for the induction of neural tube and neural crest lineages. Co-differentiation of CNS neurons or melanophores with epidermal cells can be obtained in a single well of co-cultures of VE cells (150) and a wide range of numbers of MZ cells (5 to 100). Further, reproducible differentiation of both neural lineages requires intimate association between cells from the two gastrula regions; virtually no differentiation is obtained when cells from the VE and MZ are separated in a culture well. These results indicate that the inducing signals from MZ cells for both neural tube and neural crest lineages affect only nearby ectoderm cells.