FVIII immunostaining with ab53703 in primary mouse liver cells after culture for 48 hours. (A) LSECs from hemophilia A mouse with no FVIII staining. (B) LSECs from healthy C57BL/6 mouse with FVIII as punctate green cytoplasmic dots. (C) CD11b⁺ KCs from hemophilia A mouse with no staining. (D) CD11b⁺ KCs from healthy C57BL/6 mouse with FVIII in cytoplasm. The abundance of FVIII in KCs was less than in LSECs. (E) FVIII mRNA in fractionated human BM by single-step RT-PCR: lane 1, molecular weight marker; lane 2, CD105 (mesenchymal and vasculogenic endothelial) cells; lane 3, CD33 (myeloid) cells; lane 4, MSCs; lane 5, CD133 (hematopoietic precursor) cells; lane 6, total BM MNCs; lane 7, adult human hepatocytes (Hep); lane 8, fetal human liver (FL); and lane 9, PCR mix (W). β-actin was amplified to verify RNA integrity. (F) Single-step RT-PCR for FVIII in CD34⁺ human CB cells (lane 1), total human CB cells (lane 2), BM-derived hMSCs used for transplantation studies in NOD/SCID hemophilia A mice (lane 3), and PCR mix (W; lane 4). (G) Flow cytometry of BM for abundance of CD105 cells from hemophilia A mouse, GFP transgenic donor mouse, and 2 hemophilia mice 9 months after transplantation of 10 × 10⁶ BM cells from GFP donor mice. Note CD105⁺ cell fraction in BM of hemophilia mouse and GFP⁺ donor mouse was 2% and 1.7%, respectively (R2⁺R4 gates), with GFP expression in 70% of CD105 cells in donor. After BM transplantation, GFP⁺ donor BM cells constituted 33% and 26% of CD105 cells in hemophilia A mice. (H-I) FVIII staining with ab53703 in BM-derived hMSCs in culture with no signals when FVIII antibody was omitted (H) and staining of FVIII in cytoplasm (green dots) after inclusion of FVIII antibody (I). Panels A through D and H and I, DAPI counterstain (blue); original magnification ×630.