transplantation-human-induced-pluripotent-stem-cell-derived-microglia

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

University of Yamanashi

Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y receptors (P2YR) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2YR in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2YR to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2YR expression. The number of injured hippocampal neurons was significantly higher in P2Y R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2YR KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen-glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2YR in microglia. In summary, these data suggest that microglial P2YR mediate neuroprotective effects against ischemic stress and OGD insult.

Neuroprotective effects of microglial P2Y receptors against ischemic neuronal injury.

Microglia are the resident immune cells of the brain, and play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are relatively different from mouse microglia. However, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To elucidate the in vivo characteristics of human microglia, methods have been developed to generate and transplant induced pluripotent or embryonic stem cell-derived human microglia into neonatal or adult mouse brains. However, its widespread use remains limited by the technical difficulties of generating human microglia, as well as the need to use immune-deficient mice and conduct invasive surgeries. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia and transplant them into the brain via a transnasal route in immunocompetent mice, in combination with a colony stimulating factor 1 receptor antagonist. We found that human microglia were able to migrate through the cribriform plate to different regions of the brain, proliferate, and become the dominant microglia in a region-specific manner by occupying the vacant niche when exogenous human cytokine is administered, for at least 60 days.

Transnasal transplantation of human induced pluripotent stem cell-derived microglia to the brain of immunocompetent mice.

Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca imaging from Adora2b knockout mice indicated that the A receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A receptor increased the number of excitatory synapse in developmental stage. Taken together, the A receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.

Adenosine A receptor down-regulates metabotropic glutamate receptor 5 in astrocytes during postnatal development.

Activation of astrocytes has a profound effect on brain plasticity and is critical for the pathophysiology of several neurological disorders including neuropathic pain. Here, we show that metabotropic glutamate receptor 5 (mGluR5), which reemerges in astrocytes in a restricted time frame, is essential for these functions. Although mGluR5 is absent in healthy adult astrocytes, it transiently reemerges in astrocytes of the somatosensory cortex (S1). During a limited spatiotemporal time frame, astrocytic mGluR5 drives Ca2+ signals; upregulates multiple synaptogenic molecules such as Thrombospondin-1, Glypican-4, and Hevin; causes excess excitatory synaptogenesis; and produces persistent alteration of S1 neuronal activity, leading to mechanical allodynia. All of these events were abolished by the astrocyte-specific deletion of mGluR5. Astrocytes dynamically control synaptic plasticity by turning on and off a single molecule, mGluR5, which defines subsequent persistent brain functions, especially under pathological conditions.

Article	Year
Neuroprotective effects of microglial P2Y receptors against ischemic neuronal injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism\| PubMed ID: 30334687	2019
Transnasal transplantation of human induced pluripotent stem cell-derived microglia to the brain of immunocompetent mice. Glia\| PubMed ID: 34309082	2021
Adenosine A receptor down-regulates metabotropic glutamate receptor 5 in astrocytes during postnatal development. Glia\| PubMed ID: 34339538	2021
Transient astrocytic mGluR5 expression drives synaptic plasticity and subsequent chronic pain in mice. The Journal of experimental medicine\| PubMed ID: 35319723	2022

Bijay Parajuli

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