Comprising about 40% of all cells in human brains astrocytes have long been classified as mere passive support cells. Recent work, however, has demonstrated that astrocytes play many active roles and are critical for the development and function of the central nervous system (CNS). For example, purified neurons in culture are incapable of forming synapses; instead, synaptogenesis proceeds only in the presence of astrocytes or astrocyte-secreted proteins. Astrocytes are not only important for the formation of synapses, but are also essential for the phagocytic elimination of synapses and the refinement of developing neural circuits. Since synapse formation and elimination are key cellular processes occurring during learning and memory, astrocytes are postulated to be an indispensable component in CNS plasticity. Additionally, astrocytes are required for neurotransmitter recycling, extracellular potassium homeostasis, regulation of blood flow, and providing energy substrates for neurons. Considering their central role in CNS physiology, it is not surprising that astrocyte dysfunction has been demonstrated or implicated in nearly all neurological disorders. But the extent of our understanding of astrocyte physiology in health and disease is almost entirely restricted to observations in rodent models. How primary human and murine astrocytes compare at molecular and functional levels remains largely unknown.
Observational studies from sectioned postmortem human tissues have revealed that human astrocytes are much larger and more complex than their rodent counterparts. Additionally, functional studies in organotypic cultures have revealed that calcium transients propagate faster in human astrocytes than in rodent astrocytes. More recently, transplantation of human glial progenitors into mouse brains have been shown to improve learning and memory in the chimeric mice. These observations raise questions about how rodent astrocyte physiology and function might extend to humans, and whether human astrocytes have distinct properties that make them better suited for contributing to the unique intelligence of humans. The roles astrocytes play in neurological disorders and development of effective therapeutic approaches to help human patients suffering from neurological disorders may depend on the ability to isolate functional human astrocytes.
A major hurdle in addressing these issues is the lack of a method to acutely purify human astrocytes and culture them in chemically defined conditions. Current purification methods for human astrocytes are based on a protocol developed by McCarthy and de Vellis over 30 years ago, which requires culturing dissociated nervous tissue in serum for days. Exposure to serum is sufficient to kill the majority of cells, except for a small population of astrocyte progenitor-like cells that survive and proliferate to eventually populate the culture. In vivo, however, quiescent astrocytes do not contact serum except upon injury and blood-brain-barrier break down, and in vitro exposure to serum has been shown to induce irreversible reactive changes in astrocytes. Moreover, since serum-selection methods require a group of proliferating astrocyte progenitors, these protocols do not work efficiently to purify mature astrocytes from adult human brains. Because of these limitations, the transcriptome profile of mature resting human astrocytes is unknown.
Deriving astrocytes from induced pluripotent stem cells or iPSCs is an attractive alternative for obtaining patient-derived astrocytes. There are a variety of protocols for differentiating iPSCs into astrocytes. However, without a transcriptome dataset of acutely purified primary human astrocytes, it is unclear whether iPSC-derived astrocytes closely resemble astrocytes in vivo and it is impossible to determine which differentiation protocol produces the best model for human astrocytes. Since iPSC-derived astrocytes are generated in weeks or months and human development happens over years, iPSC-derived astrocytes are a better model for fetal astrocytes than adult astrocytes. Therefore, there are additional challenges in modeling adult-onset neurological disorders, for example Alzheimer's disease, with iPSC-derived astrocytes.
To better understand the function and gene expression profiles of human astrocytes, and to provide a source of human astrocytes for therapeutic and research purposes, a method to acutely purify astrocytes from fetal and postnatal human brains and to culture these cells in chemically defined serum-free conditions is desirable.