Figure 1

Methodological schematic representations for the study. (a) Protocol for the network formation assay. RBC depleted human umbilical cord blood (UCB) cells and peripheral blood-derived mononuclear cells (PB-MNCs) were co-cultured on OP9 stromal cells (i.e. OP9 pre-conditioning) in 10-cm dishes. After 18–24 h culture, adherent cells were harvested and subjected to in vitro network formation assay. RBC depleted crude UCB cells and PB-MNCs cultured on fibronectin-coated dishes were used as control. (b) Protocol for the single cell RNA sequencing (scRNA-seq) and flow cytometry. RBC depleted crude UCB cells were used as control. (c) Protocol for intravenous administration of OP9 pre-conditioned UCB in the MCAO mouse model. Mice were randomly assigned to three groups as follows: the UCB + OP9 group (n = 11); the control group (n = 11); and the sham surgery group (n = 12) and underwent MCAO or sham surgery. Two weeks after surgery, mice in the UCB + OP9 group received 100 μL of OP9 pre-conditioned UCB (2.0 × 10⁷ cells/kg) and mice in the control group received the same amount of lactated Ringer’s solution intravenously (iv.) Next one month after cell transplantation, a series of behavioral tests were performed, and three months after cell transplantation, five mice in the UCB + OP9 group and five mice in the control group were randomly selected and subjected to the immunohistochemistry.