A New Conditionally Immortalized Human Fetal Brain Pericyte Cell Line: Establishment and Functional Characterization as a Promising Tool for Human Brain Pericyte Studies

Abstract

While pericytes wrap around microvascular endothelial cells throughout the human body, their highest coverage rate is found in the brain. Brain pericytes actively contribute to various brain functions, including the development and stabilization of the blood-brain barrier (BBB), tissue regeneration, and brain inflammation. Accordingly, detailed characterization of the functional nature of brain pericytes is important for understanding the mechanistic basis of brain physiology and pathophysiology. Herein, we report on the development of a new human brain pericyte cell line, hereafter referred to as the human brain pericyte/conditionally immortalized clone 37 (HBPC/ci37). Developed via the cell conditionally immortalization method, these cells exhibited excellent proliferative ability at 33 °C. However, when cultured at 37 °C, HBPC/ci37 cells showed a differentiated phenotype that was marked by morphological alterations and increases in several pericyte-enriched marker mRNA levels, such as platelet-derived growth factor receptor β. It was also found that HBPC/ci37 cells possessed the facilitative ability of in vitro BBB formation and differentiation into a neuronal lineage. Furthermore, HBPC/ci37 cells exhibited the typical "reactive" features of brain pericytes in response to pro-inflammatory cytokines. To summarize, our results clearly demonstrate that HBPC/ci37 cells possess the ability to perform several key brain pericyte functions while also showing the capacity for extensive and continuous proliferation. Based on these findings, it can be expected that, as a unique human brain pericyte model, HBPC/ci37 cells have the potential to contribute to significant advances in the understanding of human brain pericyte physiology and pathophysiology.

NLRP3 Is Involved in the Maintenance of Cerebral Pericytes

“…Human primary brain vascular pericytes (HBPC) were immortalized by infecting cells with tsSV40T lentiviral particles (Umehara et al, 2018). The selected immortalized HBPC clone 37 (hereafter referred to as HBPC/ci37) was used for our study.…”

“…Pericytes were cultured in a 12-well plate at 5.0 × 10 5 cells/well. Before experiments, we replaced the culture medium with serum-free pericyte medium and cultured cells at 37 • C for 3 days to facilitate cell differentiation (Umehara et al, 2018). Thereafter, pericytes were treated for 24 h with MCC950, at 0, 25, 50 and 100 nM, recombinant human IL-1β (Catalog: #201-LB; R&D Systems) at 0, 5, 10 and 50 ng/ml, or AKT Inhibitor VIII (Catalog: #124018; Sigma-Aldrich) at 0, 0.5, 1 and 5 µM.…”

Modeling blood–brain barrier pathology in cerebrovascular disease in vitro: current and future paradigms

“…Immortalized cell lines are often used for in vitro BBB modeling. The most utilized cell lines used for BECs are TY08, HMEC-1 and HCMEC/D3; for astrocytes, A735 and C6; for microglial cells, HMO6; and pericytes, HBPC/ci37 [99][100][101][102][103][104][105]. Cells can also be isolated from tumors like teratoma (NT2 cell line, for deriving neurons and oligodendrocytes) or neuroblastoma (SH-SY5Y neuronal cell line) [106,107].…”

NLRP3 maintains healthy pericytes in the brain

“…Human primary brain vascular pericytes (HBPC) were immortalized by infecting cells with tsSV40T lentiviral particles [24]. The selected immortalized HBPC clone 37 (hereafter referred to as HBPC/ci37) was used for our study.…”

“…Pericytes were cultured in 12-well plate at 5.0 × 10 5 cells/well. Before experiments, we replaced culture medium with serum-free pericyte medium and cultured cells at 37°C for 3 days to facilitate cell differentiation [24]. Thereafter, pericytes were treated for 24 hours with MCC950, at 0, 25…”