Jane Bottenstein - Profile on Academia.edu (original) (raw)

Papers by Jane Bottenstein

Cover illustration is a micrograph of a differentiating cortical neurosphere immunostained with a... more Cover illustration is a micrograph of a differentiating cortical neurosphere immunostained with antibodies for nestin (green) and propidium iodide (red); see Chapter 3 by Nakano and Kornblum.

Heparin binding factors in cns neuronal cell line conditioned medium stimulate proliferation of 0 2a glial progenitors

The Society for Neuroscience Abstracts, May 10, 1989

Proliferation of glioma cells in serum-free defined medium

PubMed, 1981

The requirement for a serum supplement to basal culture medium to support the growth and viabilit... more The requirement for a serum supplement to basal culture medium to support the growth and viability of cells in vitro has been a major hindrance to reducing and thereby controlling experimental variables. The complexity and undefined nature of serum as well as the variability of serum lots make it difficult to reproduce data and to interpret results of experiments. This paper presents preliminary results obtained with C6 glioma cell cultures in which serum was replaced by growth factors and a hormone. A brief review of the use of serum-free defined medium for the culture of tumor cells and the potential uses of defined media that are cell-type specific are also presented.

Effects of Nerve Growth Factor on Cyclic Amp Levels in Embryonic Chick Dorsal Root Ganglia Following Factor Deprivation

Journal of Neurochemistry, Jun 1, 1979

Nerve growth factor (NGF) at 10 B.U./ml produced a 5‐fold increase in the concentration of cyclic... more Nerve growth factor (NGF) at 10 B.U./ml produced a 5‐fold increase in the concentration of cyclic AMP 10min after addition to freshly excised embryonic chick dorsal root ganglia (DRG). The presence of NGF at 10 B.U./ml, but not 1 B.U./ml, over a 6 h incubation in nutrient‐free medium prevented this cyclic AMP increase when the DRG were again challenged with NGF (10 B.U./ml) after the 6 h. Incubation of DRG for 6 h at 37°C without NGF did not prevent the cyclic AMP response from occurring when NGF was presented at this time. The basal cyclic AMP concentration, however, decreased by 65–75% during the course of the 6 h incubation, and the continuous presence of NGF (10 B.U./ml) was unable to prevent this. When NGF was added to 6‐h NGF‐deprived DRG, the time for maximum cyclic AMP increase decreased from 15min to 6min with increasing NGF concentrations from 1 to 50 B.U./ml, although the relative magnitude of the cyclic AMP increase was essentially the same (approx 3‐fold) at these NGF concentrations.Attempts were made to correlate the cyclic AMP response in NGF‐deprived DRG with another rapid response resulting from administration of NGF to NGF‐deprived DRG, namely, the reactivation of hexose uptake. The cyclic AMP and permeation responses both occurred on the same time scale (5‐15 min), and both responded to increasing concentrations of NGF with a decreasing time to achieve maximal effect. A temporal relationship between cyclic AMP and membrane permeability was noted, but it could not be ruled out that it might reflect difficulties in methodology and/or inadequacy in current knowledge of the underlying mechanisms.

Developmental Brain Research, Jul 1, 1990

Responses of ohgodendrocyte/type 2 astrocyte (O-2A) ghal progenitors from neonatal rat brains to ... more Responses of ohgodendrocyte/type 2 astrocyte (O-2A) ghal progenitors from neonatal rat brains to different growth factors were studied by a new, serum-free method Enriched tertiary cultures of O-2A progenitors were produced after 6-7 days m vitro using the growth-promoting factors from the B104 CNS neuronal cell hne, heparin, and mechanical separation These cultures contained about 75-90% A2B5 + cells with less than 10% type 1 astrocytes, and the yield was 4 4 × 105 cells/brain B104 conditioned medium (CM) factors increased both O-2A progemtor number and [3H]thymidme-labehng indices after three days However, type 1 astrocyte CM was required for continued survival of enriched progemtors beyond 1 day m tertiary culture Platelet-denved growth factor (PDGF) and gha maturation factor also showed growth-promoting action, but were less effective than BIlM CM at tested doses PDGF-neutrahzmg antibodies had no effect on progenitor survival or response to B104 CM factors Thus, type 1 astrocyte-denved PDGF was not required for th~s response, B104 CM is not hkely to contain PDGF, and B104 CM factors act directly on O-2A progenitors Fibroblast growth factor, transforming growth factor fl, lnterleukln 2, epidermal growth factor, and trnodothyromne showed no growth-promoting activity, moreover, lnterleukm 2, epidermal growth factor, transforming growth factor fl, and 0 5% fetal bovine serum inhibited B104 CM action. Enriched progenitors exhibited bipotentlahty by slowly differentiating into ohgodendrocytes in serum-free medium, whereas culture in 10% fetal bovine serum increased type 2 astrocytes Thus, this new method selects or produces progemtors which are simdar to those from mature brains

Journal of Neuroscience Research, Apr 1, 1991

During development, myelin-forming oligodendrocytes and type 2 astrocytes are believed to arise f... more During development, myelin-forming oligodendrocytes and type 2 astrocytes are believed to arise from bipotential (0-2A) glial progenitors. Previously we found that conditioned medium (CM) from the B104 rat CNS neuronal cell line promotes growth of neonatal rat 0-2A progenitors in serum-free culture conditions with subsequent increases in differentiated progeny. We now report that 0-2A progenitors are present in mature rat brains and that this CM promotes the growth, motility, and bipolar morphology of these cells from 30-and 65-day-old rat brains, as shown by quantitative studies using double immunostaining and [3H]thymidine-autoradiography. In addition, the growth-promoting action of B104 CM is not neutralized by antibodies to platelet-derived growth factor, a proposed progenitor mitogen. Subsequent to the proliferation of these 0-2A progenitors, increases in oligodendrocytes and type 2 astrocytes occur. These data suggest a novel therapeutic strategy for some demyelinating diseases, e.g., multiple sclerosis, where there is a deficit in oligodendrocytes. Although it has been proposed by others that mature brain 0-2A progenitors are less proliferative and thereby incapable of adequately replenishing lost oligodendrocytes in these diseases, we present in vitro evidence for continued response of mature brain 0-2A progenitors to this neuronal cell line-derived mitogen.

Proliferation of glial-derived cells in defined media

Journal of Neuroscience Research, 1982

A serum‐free defined medium has been formulated that supports proliferation and morphologic diffe... more A serum‐free defined medium has been formulated that supports proliferation and morphologic differentiation of U‐251 MGsp human and C6‐2BD rat glioma cells. This defined medium consists of a basal medium supplemented with transferrin, fibroblast growth factor, hydrocortisone, selenium, biotin, and fibronectin (G2 medium). When U‐251 cells were plated in G2 medium on poly‐D‐lysine precoated dishes, their growth rate was 77% and final cell density was 82% of serum‐grown counterparts. The growth rate of C6 cells in G2 medium was 67% compared to cells cultured in serum supplemented medium. Although G2 medium supported the growth of human and rat glioma cells, LA‐N‐1 human neuroblastoma and WI‐38 human fibroblast cells showed no increase in cell number when grown in G2 medium compared to basal medium. A similar formulation (G3 medium), lacking fibroblast growth factor and hydrocortisone, supported the proliferation of RN‐22 rat schwannoma cells. Morphologic differences were observed between cells grown in the presence of serum and in defined media. All three glial cell lines changed from a flattened shape in serum supplemented medium to a more spherical appearance in defined medium. In addition, both U‐251 and C6 cells developed numerous processes, some reaching several cell diameters in length. These defined media will facilitate studies of the growth and differentiation of glial‐derived cells.

Developmental Biology, Jul 1, 1981

The embryonal carcinoma line C17-Si clone 1003 is multipotential in vivo. When the cells are grow... more The embryonal carcinoma line C17-Si clone 1003 is multipotential in vivo. When the cells are grown in vitro in serum-containing medium most of them remain undifferentiated, while a few differentiate into a unique morphologic type of epithelioid cell. If 1603 cells are passaged into a defined medium containing insulin, transferrin, selenium, and fibronectin they grow for six to eight generations at the same rate as in serum-containing medium. During this time, all the cells of the culture differentiate into a limited number of phenotypes with neuroepithelial and neuronal cells predominating. Differentiation could be obtained in the defined medium at relatively low cell densities. Exogenous fibronectin is required for cell attachment to the substratum, and when absent the cells form aggregates in which differentiation still occurs. Low amounts of serum added to the defined medium allow multiplication and maintenance of cells of undifferentiated phenotype and prevent differentiation into neuronal cells.

Experimental Cell Research, Oct 1, 1980

We have demonstrated in this study that we could eliminate the requirement of a serum preincubati... more We have demonstrated in this study that we could eliminate the requirement of a serum preincubation for proliferation of B104 neuroblastoma cells in defined medium. When cells were plated directly into serum-free defined medium after trypsin or EGTA detachment, they had no difftculty in adhering or remaining attached to the plastic substratum but were incapable of cell division. However, the addition of human plasma fibronectin to serum-free defined medium and precoating the tissue culture dishes with polylysine at each subculture permitted cell division to occur. Fibronectin was only required at the time of subculture and did not need to be replenished at each medium change. In addition, we have shown that clonal growth and serial subculture are possible in serum-free defined medium provided that the cell inoculum encounters the appropriate substratum. These findings are consistent with a role for fibronectin and a positively charged substratum in the growth regulation of B104 neuroblastoma cells. This completely defined culture system will be of great benefit in studying the growth regulation and differentiation of these neuronal cells.

Experimental Cell Research, 1980

Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue cu... more Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue culture dishes in Eagle's basal medium containing 10% fetal calf serum (FCS). After 48 h, which allowed adequate cell attachment, the cultures were washed with serum-free medium and then received fresh medium supplemented with 10 % FCS or serum-free defined medium (Nl), which was supplemented with insulin, transfer&, progesterone, putrescine and selenium. In addition, both media required the addition of Nerve Growth Factor (NGF). Nl medium selectively maintained the neurons and did not support proliferation or even survival of almost all non-neuronal elements (fibroblasts and Schwann cells). Survival of neurons in Nl was initially as good and eventually better than in serum-containing medium. After 6 days in Nl the cultures consisted almost entirely of neurons (>95%), which had smaller cell bodies but more extensive process formation than in serum-supplemented medium. The omission of any one of the supplements resulted in a reduction of neuron survival. The ability to generate cultures of pure neurons in a serum-free defined medium may be useful for studying (i) the role of specific hormones and growth factors normally supplied by serum in the maintenance of neurons and (ii) biochemical parameters of neurons in the absence of the substantial background due to non-neuronal elements.

Proceedings of the National Academy of Sciences of the United States of America, 1979

Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca2+ concentration

Journal of Neurobiology, 2004

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B1... more We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long‐standing observations that somal survival decreases with more‐proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine‐ or thapsigargin‐induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+‐induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 137–153, 2004

Growth Requirements of Neural Cells in Vitro

Elsevier eBooks, 1983

Publisher Summary This chapter discusses factors known to influence the survival and growth of vi... more Publisher Summary This chapter discusses factors known to influence the survival and growth of virtually all vertebrate cells as well as those of special significance for neural cells. To sustain the survival or division of most vertebrate cells in vitro , a nutrient medium must satisfy the requirements for a balanced salt solution of appropriate osmolarity, buffered hydrogen ion concentration, carbohydrate source, amino acids, vitamins, trace elements, and growth factors. A few cell types can survive in basal medium alone, but the continued survival or division of most cells still requires supplementation with serum or other undefined additives. Under normal conditions in vivo , the brain, unlike most other tissue, derives its energy almost exclusively from the oxidation of glucose. The ability to select for specific cell types from a mixed culture is a clear benefit of using defined medium, as is the suppression of fibroblast overgrowth, even in cultures where the selectivity is not absolute.

Pure astrocyte cultures derived from cells isolated from mature brain

Glia, 1988

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30‐day... more Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30‐day‐old rat brain, eventually yield cultures in MEM‐15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast‐like cells. If these cultures are switched to a serum‐free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are < 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain < 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP–/GC–. In serum‐free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.

Journal of Neuroscience Research, Jul 1, 1988

100°C treatment for 20 min, and appear to be 30-100 kilodaltons by stirred cell ultrafiltration. ... more 100°C treatment for 20 min, and appear to be 30-100 kilodaltons by stirred cell ultrafiltration. In summary, we have identified a potent source of growth-stimulating factors that produce increased numbers of glial progenitor cells and oligodendrocytes; the same conditioned medium also appears to inhibit type 1 astrocyte proliferation.

Life Sciences, Aug 1, 1978

Ia C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, ... more Ia C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, ae wall ae an induction of lactate dehydrogeaase. All of these responses were blocked by 1-propranolol, suggesting mediation by a ß-receptor. Phentolamise potentiated the NE-increased CAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of a-adrenergic receptors in C6 cells. Carbamylcholiae decreased the levels of both cyclic nucleotides, with hexamethoaium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catacholsmise-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% sad decreased cyclic AMP levels 36Z. Calcium-and magnesium-free media inhibited the norepiaephrineinduced levels of cyclic GMP and cyclic AMP respectively. Ia the rat adrenal medulla a causal relationship between an initial increase is CAMP in response to acetylcholine and the subsequent induction of

Environmental Influences on Cells in Culture

Humana Press eBooks, Nov 14, 2003

ABSTRACT The technique of culturing cells derived from the nervous system has been in use for ove... more ABSTRACT The technique of culturing cells derived from the nervous system has been in use for over 80 years, with the objective of simplifying the experimental system to provide readily manipulatable models of neural function. These preparations are useful for testing hypotheses relevant to cell adhesion, motility, survival, proliferation, longevity, and expression of cell type-specific properties. Many significant modifications of these methods have resulted over the years, as new data have emerged from cell biology and neurobiology studies (see reviews by Bottenstein, 1983a,Bottenstein, 1985,Bottenstein, 1988).

Brain Research, Sep 1, 2006

Growth factor-dependent proliferation of neuronal progenitors is an essential stage in CNS develo... more Growth factor-dependent proliferation of neuronal progenitors is an essential stage in CNS development. Although several of these growth factors have been identified, high levels of neuregulin 1 (NRG1) mRNA and protein expression in the CNS during the time of neuronal progenitor expansion suggest NRG1 growth factors may also play a key role in their proliferation. No previous studies have examined the expression of multiple NRG1 isoforms and receptors in these progenitors and their role in proliferation or apoptosis. Using a rat CNS clonal cell line with neuronal progenitor properties, we show for the first time these cells coexpress multiple NRG1 isoforms (NRGβ1, NRGβ3, CRD-NRGβ, and SMDF, but not GGF2 or any α isoforms) and all three cognate receptors (erbB2-4). We also show for the first time the presence of mRNA for all four variants of the erbB4 receptor in a single CNS cell type. Neutralizing antibody treatments suggest NRG1 isoforms and receptors are involved in proliferation but not apoptosis of these cells. This model system should be useful in future studies of the ligand specificity and function(s) of the erbB4 receptor variants.

Developmental Brain Research, Sep 1, 1989

The growth-promoting activity of conditioned medium (CM) from the B104 CNS neuronal cell line was... more The growth-promoting activity of conditioned medium (CM) from the B104 CNS neuronal cell line was studied in glial cultures from neonatal rat brain. This CM at 33% (v/v; 8-12/zg protein/ml) produced large numbers of oligodendrocytes and multipolar glial progenitors after an 8 to 12-day treatment. At all times studied, cells of the oligodendrocyte/type 2 astrocyte (O-2A) lineage were increased due to CM-treatment, while type 1 astrocytes, microglia, and other cell types were not. Furthermore, we observed a large decrease in the percentage of oligodendrocytes in the O-2A lineage, suggesting a delay in differentiation of the progenitors. By 8 days in vitro (DIV), dose-dependent increases in numbers of galactocerebroside (GalC)-positive cells (oligodendrocytes) and A2B5-positive cells (immature oligodendrocytes and glial progenitors) occurred, In contrast, at 4 DIV only A2B5-positive cells were increased in a dose-dependent manner. The latter cells can differentiate primarily into oligodendrocytes or type 2 astrocytes depending on the culture conditions. Complement lysis studies confirmed that the A2B5-positive, but not the GalC-positive, population at 4 DIV was required for increases in oligodendrocytes to occur by 8 DIV. The [3H]thymidine labeling Index of the A2B5-positive population also increased in response to CM in a dose-dependent manner, but the GalC-positive labeling index showed only small increases at 4 DIV and none at later times. Our results suggest that the delayed differentiation coupled with the selective stimulation of the bipotential glial progenitors produces the large increases in numbers of oligodendrocytes observed at 8-12 DIV.

Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 1986

I have defined the basic requirements for the proliferation of cell lines expressing oligodendroc... more I have defined the basic requirements for the proliferation of cell lines expressing oligodendrocyte properties and for the survival of galactocerebroside-positive oligodendrocytes derived from neonatal rat brains. Conventional serum-containing medium can be replaced by 01 medium, a chemically defined medium supplemented with insulin, transferrin, sodium selenite, and biotin. Thyroid hormone is not required. When cells are plated directly into 01 medium, the substratum has to be modified by precoating with polylysine and adding fibronectin to the medium prior to the cells. Both cell lines and brain cells can be subcultured numerous times in 01 medium without initial culture in serum-containing medium. Brain cultures can be maintained in 01 medium for several months and contain a significantly higher percentage of mature oligodendrocytes, a lower number of astrocytes, and no fibroblasts as compared to cells maintained in serum-containing medium.