Figure 6.

In vitro and in vivo analysis of the supporting activity of MSCs on GE mobilized PB (GE-mPB) HSPCs

(A) Schematic representation of the in vitro and in vivo experiments using GE-mPB CD34⁺ cells. BM-MSCs were expanded for 72 h before co-culture with mPB HSPCs. Thereafter, mPB CD34⁺ cells were pre-stimulated in co-culture with BM-MSCs in GE medium conditioned from BM-MSCs and similarly recovered upon gene editing. GE-mPB HSPCs were transplanted into NSG mice after 24 h recovery, while GE-HPSCs were maintained in culture with BM-MSCs for 72 h for the in vitro analysis. mPB HSPCs GE according to our standard protocol were used as controls for the transplantation experiments at 24 h after gene editing and for the in vitro analysis after 72 h of culture. (B and C) Total cell counts (B) and phenotypic analysis (C) of GE-mPB recovered in culture 72 h after gene editing in the presence of MSCs (mPB CD34⁺ GE + MSCs). mPB HSPCs GE according to our standard protocol conditions were used as control (mPB CD34⁺ GE). The absolute number of phenotypically primitive GE-mPB HSPCs is reported in (D). BAX, p21, and IL-8 expression analysis is reported in (E), whereas evaluation of apoptosis in GEmPB HSPCs recovered in culture 72 h after gene editing in the presence (mPB CD34⁺ GE + MSCs) or absence (mPB CD34⁺ GE) of an MSC feeder is reported in (F). (G) Percentage of human cell engraftment (%hCD45⁺) on total live cells in the PB of NSG mice transplanted with the outgrown of 0.5 × 10⁶ mPB CD34⁺ cells 24 h after GE in the presence of MSCs (mPB CD34⁺ GE + MSCs) or according to our standard protocol (mPB CD34⁺ GE) at 6 and 12 weeks after transplantation. (H) Analysis of human engraftment in the BM of transplanted mice at sacrifice (12 weeks). (I) Percentage (left panel) and absolute number (right panel) of HDR GE cells (GFP+) in the BM of transplanted mice at 12 weeks after transplantation. (J) Percentage of human cells positive for CD45 and CD34 engrafted on total live cells based on physical parameters in the BM after the transplantation of mPB CD34+ cells GE in the presence of MSCs (mPB CD34⁺ GE + MSCs) or according to our standard protocol (mPB CD34⁺ GE). For all plots, individual data points represent an animal. p values were determined by Mann-Whitney test (∗p ≤ 0.05; ∗∗p ≤ 0.001).