Safety and efficiency of hypoxic culture. (A) Karyotyping analysis shows hypoxic MSCs have normal karyotype. (B) For in vivo bone formation, cells were delivered in ceramic cube and induced in osteogenic medium for one week followed by transplantation under beneath the dorsal skin of NOD-SCID mice for 4 weeks. Mallory trichrome staining (left panel) shows hypoxic cells increase in collagen synthesis. Micro-CT (right top panel) shows hypoxic cells increase in trabecular formation. DEXA (right bottom panel) shows hypoxic cells increase in bone mineral density. (C) For in vivo fat formation, cells were mixed with basic FGF and transplanted in NOD-SCID mice for 4 weeks. Oil Red O staining shows hypoxic cells increase in the accumulation of fat droplets. (D) For in vivo cartilage formation, cells were encapsulated in alginate beads and induced in chondrogenic medium for one week followed by transplantation into NOD-SCID mice for 4 weeks. Alcian Blue staining and immunohistochemistry for type II collagen demonstrate hypoxic cells increase in the synthesis of proteoglycan and type II collagen. Bar = 50 μm.