MS4A3 enhances GM-CSF/IL-3-induced receptor endocytosis, signaling, and differentiation in primary CML CD34⁺ cells. (A) Experimental outline of primary CML sample analyses. Both Lv-MS4A3-OE and Lv-dox-shMS4A3 carry GFP to mark transduced cells. GFP^- cells are untransduced internal assay controls in the same samples, receiving the same treatment and analyses. Untransduced cells in both groups are combined for analysis. BIT supplement (BIT9500) contains BSA, human insulin, and human transferrin-Fe. StemSpan (CC100) is a cytokine cocktail for HSC and progenitor expansion. (B,C) Flow cytometry analysis of GM-CSF-induced CD116 endocytosis in CML CD34⁺ cells (n = 3). (D-E) Intracellular phospho-kinase staining of CML CD34⁺ cell cultures after GM-CSF and IL-3 stimulation for 30 minutes (n = 6). (F) Relative abundance of CD34⁺ and CD34^- cells in the single cytokine pulsing differentiation assay (n = 3). (G) Colony formation of CML CD34⁺ cells with or without GM-CSF/IL-3-induced differentiation (n = 3). (H) Illustration of the nanoparticle targeting strategy. (I) Size distribution of the manufactured MS4A3 nanoparticles. Sizes are calculated from a standard curve made by a series of microbeads of known sizes. (J) Contour plot of MS4A3 nanoparticles (EGFP⁺) carrying CD34 ligand, CD62L. (K) Delivery and retention of MS4A3-EGFP by CD62L-coated nanoparticles, as tested in CD34⁺ Kasumi-1 cells. (L) Experimental design for testing the effect of nanoparticle-based MS4A3 delivery. (M,N) Proportions of various cell types in stem cell cultures with indicated treatments. (O-P) Flow cytometry detection of CD34 level on groups of cells after indicated treatments. (Q-R) Colony-forming capability of stem cells after indicated treatments.