In February 2018, the top journal of the Nature Publishing Group, Nature Neuroscience, published a research paper led by Peng Bo's team. The paper successfully unraveled the mystery of the cellular origin of microglial repopulation in the brain, ending the major debate on whether precursor cells of microglia exist in the adult mammalian central nervous system (CNS). This study holds significant scientific value for research on CNS regeneration and the maintenance of microglial homeostasis.

The Astonishing Phenomenon of Microglial Repopulation and the Major Controversy

The regenerative capacity of the adult mammalian CNS, including the brain, retina, and spinal cord, is extremely limited. For a long time, it was believed that damage to the CNS could not be repaired. Research has shown that only two types of stem cells (or precursor cells) exist in the brain: neural stem cells (NSC) and oligodendrocyte precursor cells (OPC). The former can differentiate into neurons and astrocytes, while the latter can differentiate into oligodendrocytes. However, due to the intrinsic characteristics of CNS cells and the constraints of the external environment, their differentiation capacity is limited. They cannot produce enough cells to repair the damaged CNS during neurodegenerative diseases, such as traumatic brain injury, Alzheimer's disease, and Parkinson's disease. Therefore, brain damage in adult mammals was considered irreparable.

However, in 2014, researchers from the University of California, Irvine, discovered an astonishing phenomenon of cellular regeneration in the mouse brain, rewriting the understanding of cellular regeneration in the brain. The team led by Kim Green almost completely depleted microglia in the brain (>99%) by specifically inhibiting the colony-stimulating factor 1 receptor (CSF1R) . After stopping the inhibition and a brief recovery period, new microglia appeared in the brain. These repopulated microglia rapidly colonized the entire brain and fully restored to their original state (such as density, distribution, and morphology) within about a week . This was the first time that large-scale cellular regeneration had been observed in the CNS of adult mammals, a process termed microglial repopulation. The research team speculated through histological immunostaining that the repopulated microglia might be derived from a type of cells in the brain that transiently express Nestin. This was considered the third type of stem cells discovered in the adult mammalian CNS, in addition to neural stem cells and oligodendrocyte precursor cells. During development, the original microglia originate from the yolk sac in the embryonic period, while other cells in the CNS originate from the ectoderm . Nestin cells are believed to have characteristics of ectoderm-derived stem cells, suggesting that these stem cells have a high potential for differentiation across lineages. Due to their high differentiation potential and astonishing differentiation speed, scientists believed that it might be possible to regulate these cells to differentiate into other cell types in the brain, which is of great significance for CNS injury repair and regenerative medicine. Therefore, this discovery received widespread attention upon publication, with the paper being cited more than 100 times per year.

However, behind this significant discovery lies a major controversy regarding the origin of repopulated microglia: First, whether cross-lineage differentiation can occur in the mature CNS. If repopulated microglia are derived from Nestin-positive cells, it would imply that ectoderm-derived cells can differentiate into other lineages, which is extremely rare in the adult mammalian CNS. Second, whether repopulated microglia originate from the differentiation of blood cells. During neuroinflammatory responses, a small number of blood cells can cross the blood-brain barrier and differentiate into microglia-like cells. Previous studies on the origin of repopulated microglia did not fully exclude this pathway. Third, repopulated microglia could also originate from the proliferation of residual microglia. According to Kim Green's team, a small number of microglia remain in the brain (about 1%), and these residual microglia could proliferate to form repopulated microglia, a possibility that could not be completely ruled out. Therefore, there is a major debate in the academic community regarding the origin of repopulated microglia in the brain.

Multiple Lineage Tracing Techniques Identify the Origin of Repopulated Microglia

In the research paper titled "Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion" published in Nature Neuroscience, Peng Bo's team first used rigorous multiple lineage tracing (fate mapping or lineage tracing) techniques to systematically elucidate the origin of repopulated microglia. First, the team used parabiosis to exchange the blood between wild-type mice and GFP (green fluorescent protein)-carrying mice to investigate the possible hematogenous origin of repopulated microglia. The team found that in all brain regions explored, no repopulated microglia originated from blood cells, thus excluding the hematogenous origin hypothesis of repopulated microglia.

Subsequently, the research team used the inducible lineage tracing transgenic mouse Nestin-CreER::Ai14 to label all Nestin cells. In this experiment, the recombinase CreER would excise the stop signal in front of the reporter gene tdTomato in Nestin cells under the induction of tamoxifen. Thus, these cells and their differentiated progeny would permanently express the tdTomato reporter protein. However, surprisingly, no repopulated microglia were found to carry the tdTomato reporter protein, indicating that these cells did not originate from the previously speculated Nestin-positive cells. Next, the team used multiple transgenic mouse tools to perform lineage tracing on the major ectoderm-derived cells in the brain (including astrocytes, neural stem cells, oligodendrocyte precursor cells, and various neurons) and found that none of these cells were the precursors of repopulated microglia. Instead, through the ingeniously designed Cx3cr1-CreER::Ai14 lineage tracing technique, the team discovered that all repopulated microglia originated from the proliferation of residual endogenous microglia (<0.9%) in the CNS. During this process, the newly generated microglia transiently expressed Nestin protein, but their cell fate did not change. Thus, Peng Bo's team used multiple lineage tracing techniques to overturn the previous conclusion that repopulated microglia originated from Nestin-positive precursor cells and found that repopulated microglia entirely originated from the proliferation of residual microglia. This phenomenon is the fastest cell proliferation observed in the adult mammalian CNS to date. In addition, Peng Bo's team also elucidated the possible mechanism of cell repopulation through single-cell RNA sequencing and discovered the high functional similarity between repopulated microglia and endogenous microglia through whole-brain RNA sequencing.

The paper was selected as one of the top five neuroimmunology advances in 2018 by Nature Reviews Immunology.