Graduate Thesis Or Dissertation
 

Nonsenecent serum-free mouse proastroblasts : extended culture, growth responses in vitro, and application to the culture of human embryonic astrocytes

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/m326m6295

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  • Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. I used the technique of serum-free cell culture to develop a serum-free mouse embryo (SFME) cell line in which serum was replaced by a set of defined supplements. SFME cells, cultured in a nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin, have maintained a diploid karyotype with no detectable chromosomal abnormalities for more than 200 generations. The cells did not undergo growth crisis and remain in culture today. SFME cells were dependent on EGF for survival and were reversibly growth inhibited by serum or platelet-free plasma. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acid protein (GFAP), a specific marker for astrocytes, identifying SFME cells as proastroblasts. Following the derivation of SFME cells my research focussed on (1) defining more precisely the growth response of SFME cells to medium supplements, (2) investigating the relationship between the nonsenescent nature of SFME cells and their responses to serum and EG1., and (3) applying the serum-free cell culture methods to the multipassage culture of human embryonic astrocytes. SFME cells in serum-containing medium arrested in the G1 phase of the cell cycle with greatly reduced DNA replication activity. A portion of the inhibitory activity of serum was extracted by charcoal, a procedure that removed steroid and thyroid hormones. However, the effect of serum on untransformed SFME cells could not be prevented by addition of antiglucocorticoid, and ras-transformed clones of SFME cells, which are not growth inhibited by serum, retained inhibitory responses to glucocorticoid and thyroid hormone T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are involved. SFME cell death resulting from EGF deprivation exhibited characteristics associated with apoptosis or programmed cell death. Ultrastructural analysis showed cells became small and vacuolated, with pyknotic nuclei. The cultures contained almost exclusively G1- phase cells. Chromatin exhibited a pattern of degradation into oligonucleosome-length fragments generating a regularly spaced ladder. I applied the serum-free approach used to derive SFME cells to the multipassage culture of human embryonic astrocytes. Cells were cultured in nutrient medium supplemented with insulin, transferrin, EGF, HDL, fibronectin, basic fibroblast growth factor and heparin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized GFAP.
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