Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system

Abstract

The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.Electronic supplementary materialThe online version of this article (doi:10.1007/s13365-016-0499-3) contains supplementary material, which is available to authorized users.

Human microglia and astrocytes express cGAS-STING viral sensing components

“…The human microglial cell line, hμglia, was a kind gift from Dr. Jonathan Karn (Case Western Reserve University). These cells were derived from primary human cells transformed with lentiviral vectors expressing SV40 T antigen and hTERT, and have been classified as microglia due to their microglia-like morphology, migratory and phagocytic activity, presence of the microglial cell surface markers CD11b, TGFβR, and P2RY12, and characteristic microglial RNA expression profile [16]. This cell line was maintained in Dulbecco’s Modified Eagle Medium supplemented with 5% FBS and penicillin/streptomycin.…”

The human microglial HMC3 cell line: where do we stand? A systematic literature review

“…In this respect, human microglial cell lines allow to overcome this problem and can be considered a valuable experimental model. Several cell lines have been generated in different laboratories [17,18], including more recent lines derived from adult brain tissue [19]. All these cell lines were developed through SV40 immortalization of human primary microglial cells, whereas a fraction of SV40-immortalized adult microglial cells were further engineered to express the human telomerase reverse transcriptase (hTERT) and reduce the proliferation rate [19].…”

“…Several cell lines have been generated in different laboratories [17,18], including more recent lines derived from adult brain tissue [19]. All these cell lines were developed through SV40 immortalization of human primary microglial cells, whereas a fraction of SV40-immortalized adult microglial cells were further engineered to express the human telomerase reverse transcriptase (hTERT) and reduce the proliferation rate [19]. To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [17,[20][21][22] and the "Immortalized Human Microglia -SV40", developed and distributed by Applied Biological Materials (Vancouver, Canada) [23][24][25][26][27][28].…”

LPS-induced TLR4 neuroinflammatory signaling in CHME-5 microglial cells

“…LPS: lipopolysaccharide; TLR4: tolllike receptor 4; RT-PCR: real-time polymerase chain reaction; US: unstimulated; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis Interestingly, a recent publication [25] suggested that CHME-5 cells are of rat origin, not human. In the study, they performed genotyping by macrosatellite a na lys i s, a n d i nve st i g ate d t h e CYCT1 g e n e expression in CHME-5 cells using both human and rat primers, and showed rat CYCT1 gene expression, but not the human counterpart in these cells [25] . The CHME-5 cell line currently being used in numerous labs is apparently of non-human origin, but to our knowledge this has yet to be independently verified.…”

“…Conversely, during our review of the literature we identified an article characterizing primary human microglia in which data revealed large inconsistencies in microglial and inflammatory genes between primary human microglia and human microglial cell lines, including CHME-5 cells [26] . Knowing what we do now about the non-human origin of CHME-5 cells from Garcia-Mesa et al [25] 2017, the inability to detect gene expression might have been due to the use of human primers. Taking this into consideration, we characterized TLR4 neuroinflammatory signaling in CHME-5 cells, as a rat cell line, validated that these cells are not of human origin, and demonstrated that CHME-5 cells remain a viable tool to study microglial-like inflammatory responses.…”