The potential use of bone marrow stromal cells (BMSCs) for tissue engineering and stem cell-based biotechnology awaits an efficient protocol that guides adult stem cells to a prescribed terminal differentiation. Recently, ectoderm-derivative neural cells have been generated from BMSCs, raising the question of whether adult stem cells differentiate by induction of previously unexpressed lineage-specific genes or by down-regulation of constitutively expressed lineage-inappropriate genes. Objectives: To characterize undifferentiated BMSCs and examine how their phenotypes develop during osteogenic/mesodermal and neurogenic/ectodermal differentiation. Methods: BMSCs isolated from adult mice were cultured for 6 days in non-induced medium (control medium: FBS/alpha-MEM), osteogenic-induced medium (dexamethasone,
b-glycerophosphate and ascorbic acid/control medium), and neurogenic-induced medium (B27 and BDNF/Neurobasal medium). Phenotypic profiles of the cells under each condition were determined by RT-PCR analysis, immunocytochemistry, whole-cell patch-clamp recording, and extracellular matrix (ECM)/bone related genes specific microarray analysis. Results: Non-induced BMSCs strongly expressed genes indicative of both neuronal (
b-tubulin III, nestin and NCAM) and osteogenic (osteocalcin, osteopontin and alkaline phosphatase) phenotypes. In addition, they expressed neural marker proteins (tark A and
b-tubulin III) and showed a slight voltage-activated inward membrane current. These basic properties of ectodermal lineages were enhanced when cells underwent neurogenic differentiation but disappeared during osteogenic differentiation. Microarray data showed a distinct trend toward up-regulation of ECM/bone related genes in osteogenic BMSCs when compared to non-induced BMSCs, while showing a down-regulation trend in neurogenic BMSCs. Conclusion: These results suggest that BMSCs are not 'naive', but in fact express a mixture of lineage-specific genes as a constitutive phenotype and differentiate by pruning a large repertoire of phenotypes down to lineage specific characteristics. For tissue engineering applications, it may therefore be important to lead stem cells to the targeted end point not only by up-regulating tissue phenotype sensitive genes but also by suppressing tissue phenotype non-sensitive genes.