A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype

Prah, Jude; Winters, Ali; Chaudhari, Kiran; Hersh, Jessica; Liu, Ran

doi:10.1016/j.jneumeth.2019.03.013

Cited by 31 publications

(49 citation statements)

References 52 publications

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“…Primary cell cultures are useful tools to understand astrocytic and microglia functions, although there are concerns about how these cultures accurately mirror events in vivo due to the presence of FBS in the media (Foo et al, 2011; Prah et al, 2019; Y. Zhang et al, 2016). FBS contains growth factors, proteins and vitamins that can alter cell biology and phenotype (Aswad, Jalabert, & Rome, 2016), and mimic a more inflammatory profile.…”

Section: Discussionmentioning

confidence: 99%

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation

Lanfranco

Sepulveda

Kopetsky

et al. 2021

Glia

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Neuroinflammation is a common feature in neurodegenerative diseases, modulated by the Alzheimer's disease risk factor, apolipoprotein E (APOE). In the brain, apoE protein is synthesized by astrocytes and microglia. We examined primary cultures of astrocytes and microglia from human APOE (E2, E3, and E4) targeted‐replacement mice. Astrocytes secreted two species of apoE, whereas cellular apoE consisted of only one. Both forms of secreted astrocytic apoE were bound during glycoprotein isolation, and enzymatic removal of glycans produced a convergence of the two forms of apoE to a single form; thus, the two species of astrocyte‐secreted apoE are differentially glycosylated. Microglia released only a single species of apoE, while cellular apoE consisted of two forms; the secreted apoE and one of the two forms of cellular apoE were glycosylated. We treated the primary glia with either endogenous (TNFα) or exogenous (LPS) pro‐inflammatory stimuli. While LPS had no effect on astrocytic apoE, APOE2, and APOE3 microglia increased release of apoE; APOE4 microglia showed no effect. APOE4 microglia showed higher baseline secretion of TNFα compared to APOE2 and APOE3 microglia. TNFα treatment reduced the secretion and cellular expression of apoE only in APOE4 astrocytes. The patterns of apoE species produced by astrocytes and microglia were not affected by inflammation. No changes in APOE mRNA were observed in astrocytes after both treatments. Together, our data demonstrate that astrocytes and microglia differentially express and secrete glycosylated forms of apoE and that APOE4 astrocytes and microglia are deficient in immunomodulation compared to APOE2 and APOE3.

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Section: Discussionmentioning

confidence: 99%

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation

Lanfranco

Sepulveda

Kopetsky

et al. 2021

Glia

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“…However, it is worth noting that also in those approaches, FBS was used in some purification steps of the astrocyte cultures. Recently, a method using a FBS-free, chemically defined medium containing fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF) was shown to promote the growth of mice-cultured astrocytes with morphological and biosynthetic features similar to those of in vivo astrocytes and characterized, compared to untreated cells, by a process-bearing morphology and an increase of glutamate uptake [ 70 ]. Of note, even though FGF and EGF are present in G5 cocktail, compared to untreated astrocytes, 12–16 DOT G5 astrocytes displayed a slight decrease in expression of the principal glutamate transporter GLT-1 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro

Formaggio

Fazzina

Estévez

et al. 2021

Pflugers Arch - Eur J Physiol

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The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promote the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype, which develops gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl−) and potassium (K+) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a large increase in the expression of the inward rectifier Cl− and K+ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.

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“…3 a). This phenotype typically defines a resting state (Prah et al 2019 ). Astrocytes grown with a medium containing 2 mM glucose showed hypertrophic soma and processes (Fig.…”

Section: Resultsmentioning

confidence: 99%

Long-Term Glucose Starvation Induces Inflammatory Responses and Phenotype Switch in Primary Cortical Rat Astrocytes

et al. 2021

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Astrocytes are the most abundant cell type in the brain and crucial to ensure the metabolic supply of neurons and their synapse formation. Overnutrition as present in patients suffering from obesity causes astrogliosis in the hypothalamus. Other diseases accompanied by malnutrition appear to have an impact on the brain and astrocyte function. In the eating disorder anorexia nervosa (AN), patients suffer from undernutrition and develop volume reductions of the cerebral cortex, associated with reduced astrocyte proliferation and cell count. Although an effect on astrocytes and their function has already been shown for overnutrition, their role in long-term undernutrition remains unclear. The present study used primary rat cerebral cortex astrocytes to investigate their response to chronic glucose starvation. Cells were grown with a medium containing a reduced glucose concentration (2 mM) for 15 days. Long-term glucose starvation increased the expression of a subset of pro-inflammatory genes and shifted the primary astrocyte population to the pro-inflammatory A1-like phenotype. Moreover, genes encoding for proteins involved in the unfolded protein response were elevated. Our findings demonstrate that astrocytes under chronic glucose starvation respond with an inflammatory reaction. With respect to the multiple functions of astrocytes, an association between elevated inflammatory responses due to chronic starvation and alterations found in the brain of patients suffering from undernutrition seems possible.

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A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype

Cited by 31 publications

References 52 publications

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation

Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro

Long-Term Glucose Starvation Induces Inflammatory Responses and Phenotype Switch in Primary Cortical Rat Astrocytes

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