Generation and Characterization of Immortalized Mouse Cortical Astrocytes From Wildtype and Connexin43 Knockout Mice

Abstract

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1L1 (ALDH1L1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

Connexins Signatures of the Neurovascular Unit and Their Physio-Pathological Functions

“…The high capability of endothelial cells to dynamically modulate their Cxs expression profile and the permeability of the channels they are forming is significant. Indeed, even if no direct contact between astrocytes and endothelial cells occurs in the NVU, these cells can interact in an in vitro coculture system [ 57 , 118 , 119 , 120 ]. The capability of endothelial and glial cells to effect such plastic changes, shaped the idea that they are able to modulate intercellular communication, ion homeostasis, and paracrine mediators release in a context dependent manner [ 113 , 114 , 115 ].…”

Astrocytes sense glymphatic-level shear stress through the interaction of sphingosine-1-phosphate with Piezo1

“…At 14 days, flasks were shaken at 180 rpm for 12-24 h in an incubated shaker (Ecotron HT) to remove residual tissue and microglia. 43 , 44 Astrocytes were cultured up to passage 3 for use in experiments.…”

Cooperative and competitive regulation of the astrocytic transcriptome by neurons and endothelial cells: Impact on astrocyte maturation

“…To study astrocyte/endothelial cell interactions, the mouse brain endothelioma cell line bEND.3 was used (American Type Culture Collection #CRL‐2299, RRID: CVCL_0170). We previously showed that this cell line mimics the effects of primary brain endothelia on GLT1 expression (Lee et al, 2017), and this cell line has been used to study the mechanisms that control tight junction formation (Cibelli et al, 2021; Pu et al, 2022; Sasson et al, 2021; Stamatovic et al, 2009). In addition, this cell line has the advantage of being a homogeneous population of cells.…”