NG2+ and Tbx18+ pericyte subtypes exhibit distinct reprogramming potency following ischemic stroke

Increasing evidence shows that brain pericytes can acquire multipotency to produce multi-lineage cells following injury. However, pericytes are a heterogenous population and it remains unknown whether there are different potencies from different subsets of pericytes in response to injury. Here, using single-cell RNA-sequencing (scRNA-seq) and immunohistochemistry analysis following ischemic stroke, we showed that NG2+ pericyte subset expressed very strong neural reprogramming potential to produce newborn neurons, while Tbx18+ pericytes displayed strong multipotency to produce endothelial cells, fibroblasts, and microglia but with minimal neural reprogramming potency. In addition, we mimicked ischemic stroke in vitro and developed an NG2+ pericyte neural reprogramming culture model. Here we discovered that AMP-dependent kinase (AMPK) modulators facilitated pericyte to neuron conversion by modulating Ser436 phosphorylation status of CREB-binding protein (Cbp), a histone acetyltransferase, which coordinated an acetylation shift between the non-histone substrate (Sox2) and the histone substrate (H2B) and modulated Sox2 nuclear-cytoplasmic trafficking during reprogramming/differentiation process. Finally, we identified that sequential treatment of compound C (CpdC) and metformin, AMPK inhibitor and activator respectively through reprogramming/differentiation process, robustly facilitated the conversion of human pericyte into functional neurons via transient pericyte-derived neural stem cell population. We performed scRNA-seq using ET-1/L-NAME stroke-injured cerebral cortical tissues isolated from two pericyte lineage tracing mice (NG2-Cre-ERT2/Ai14-flx and Tbx18-CreERT2/Ai14-flx) that received intracerebral injections of ET-1/L-NAME (or saline) 3 days prior to scRNA-seq. Both transgenic mice were also treated with tamoxifen 7 days before injury to enable lineage tracing of Tbx18-Ai14+ and NG2-Ai14+ cells. Three groups of Tbx18-Ai14+ and NG2-Ai14+ cells from 1) no injury (naïve), 2) physical injury (saline), and 3) ischemic injury (ET-1/L-NAME) were Fluorescence-activated Cell (FAC) sorted for tdT (Ai14)+/DAPI- live cells to perform scRNA-seq.