Retinoic acid- and TGF-β1-preconditioned hBM-MSCs show distinct reparative profiles after transplantation in Busulfan-induced testicular injury

Non-obstructive azoospermia (NOA) is a severe male-infertility phenotype characterized by intrinsic testicular failure and impaired spermatogenesis. This study compared retinoic acid (RA)- and transforming growth factor-beta 1 (TGF-β1)-preconditioned human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in a busulfan-induced mouse model of testicular injury. hBM-MSCs were characterized by plastic adherence, spindle-shaped morphology, immunophenotyping, and adipogenic/osteogenic differentiation, then preconditioned with RA (2, 5, or 10 µM) or TGF-β1 (5, 10, or 20 ng/mL) for 21 days. Busulfan-treated BALB/c mice were assigned to eight groups (n = 5/group), and preconditioned cells were transplanted four weeks after injury. Histology, modified Johnsen score, spermatogenic indices, seminiferous morphometry, testicular cell counts, epididymal sperm parameters, human-specific Alu-qPCR, and C-Kit, DAZL, OCT4, Tex18, Piwil2, and Mvh transcript expression were evaluated eight weeks after transplantation. Busulfan induced tubular atrophy, germ-cell depletion, impaired sperm-related endpoints, and downregulation of spermatogenesis-associated transcripts. Both preconditioned hBM-MSC products partially attenuated these abnormalities compared with busulfan-only injury. RA-preconditioned hBM-MSCs, particularly at 10 µM, showed the strongest pro-spermatogenic profile, whereas TGF-β1-preconditioned hBM-MSCs showed a comparatively niche-oriented epithelial and luminal recovery pattern. Human Alu-qPCR indicated donor-derived DNA persistence within testes but not viable donor-cell integration or donor-derived spermatogenesis. Because a naïve, non-preconditioned hBM-MSC group was not included, whether unprimed hBM-MSCs would produce similar, lesser, or different effects remains unknown. These findings define distinct reparative profiles, not validated molecular pathways or proven fertility restoration.