This study describes the one-step isolation and expansion of marrow stromal cells (MSCs) directly onto the implantable nanofibrous scaffolds. Coverslips were first coated with either aligned or random configurations of poly L,D lactic acid, poly lactic-glycolic acid, and poly-epsilon-caprolactone and then seeded with fresh bone marrow aspirate. Colony-forming units were quantified and the differentiation capacities of the recovered cells were explored. Further optimization was provided by exploring the impact of hyperoxic (21% O(2)) and physiologically approximate (2% O(2)) on cell recovery. Aligned nanofibers in 2% O(2) were identified as being superior for isolation of MSCs. Isolated cells formed colonies following the direction of nanofibers, indicating potential for guided tissue regeneration. The isolated MSCs demonstrated retention of multipotency. These findings offer a rapid, cost-effective method of producing a stem-cell-seeded scaffold for regeneration of multiple tissue types.