Generation of High Passage and Immortalized Human Microglia Cell Lines

Microglia are the resident immune cells of the central nervous system of myeloid lineage with diverse functions. The pro-inflammatory activation of microglia in neurodegenerative disorders and in response to trauma is well-established. Recent findings suggest that microglia also have normal physiological functions in the healthy brain. A great deal of what has been learned regarding microglia biology and function is based on in vitro studies the overwhelming majority of which used cells isolated from the rodent brain. However, higher anatomical and functional complexity of the human brain and species differences in microglia response and function make imperative the use of human microglia to ascertain that the results obtained are applicable to man. Furthermore, investigation of microglia function in the adult brain, in which many inflammatory and anti-inflammatory microglia responses occur, requires use of human microglia from adult brains. Microglia isolated and cultured from embryonic human brain show substantial proliferative capacity. However, it had not been possible to induce similar proliferation of microglia isolated from adult human brains. Thus, while methods for isolation of microglia from adult postmortem human brains exist, they allow only use of a limited quantity of microglia isolated and cultured from each case due to low levels of proliferation. In preliminary attempts, we have developed a method that allows culturing microglia isolated from adult postmortem human brains to relatively high passage. Limited preliminary testing indicated that human microglia may maintain their phenotype in high passage cultures. Based on our preliminary observations, the proposed work will attempt to generate microglia cultures of higher passage and to extensively characterize microglia phenotypes and response to different stimuli at different passages. We will also generate and extensively characterize immortalized human microglia cell lines using a well-established protocol that utilizes human telomerase reverse transcriptase. The specific aims of the proposed work will test the following hypotheses: Aim 1 - Microglia cultured from normal adult human brains of various ages and from brains of individuals with neurodegenerative disorders will proliferate to high passage in vitro. Aim 2 - High passage human microglia will maintain their phenotype, including response to various stimulants. Aim 3 - Human microglia will be successfully immortalized, and immortalized human microglia will maintain their phenotype and response to various stimulants. Successful generation and characterization of high passage and immortalized human microglia cultures will provide the scientific community with new and reliable tools for in vitro mechanistic studies of human microglia function in health from childhood to old age, and in disease, including our own studies of microglia function in Alzheimer’s disease. High yield and well characterized human microglia cultures will be necessary to carry forward many such studies. These cells could also be used to identify drugs that can regulate microglial functions in normal and diseased brain.