UM171 enriches CD14⁺/CD31⁺monocyte subset with high FVIII expression capacity. (A-B) FVIII expression in CD14⁺ monocytes differentiated from CD34⁺ cells after 14 days of monocyte-LTC (mo-LTC) assay. (A) An example of transduction efficiency and logical gating strategy for UT vs 100 MOI transduced CD34⁺ cells differentiated into CD14⁺ cells. (B) Collective analysis of FVIII expression in CD14⁺ monocytes differentiated from UT and transduced CD34⁺ cells after 14 days of mo-LTC assay (n = 3, mean). (C) A phase-contrast image (original magnification ×20) of adherence, elongation, and cell features after 7 days of the e-LTC assay (left); examples of total CD45⁺/CD14⁺/CD31⁺ cells after 14 days of the e-LTC assay, and FVIII expression in the same cells derived from UT and MOI 100 transduced CD34⁺ cells (middle); and cumulative expression of FVIII in CD45⁺/CD14⁺/CD31⁺ cells after e-LTC assay (n = 3, mean) (right). (D) FC plots and cumulative data showing CD45⁺/CD14⁺/CD31⁺ enrichment after 10 days of the CD34⁺ culture in HSC-cytokine medium with or without UM171 (n = 6, mean ± SEM) (left and middle). FVIII expression in transduced and UT CD34⁺ cells and differentiated CD14⁺/CD31⁺ cells after 7 to 10 days culture with UM171 (n = 6, mean ± SEM) (right). e-LTC, endothelial-long term culture; FC, flow cytometry; mo-LTC, monocyte-long term culture.