Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFRα+Sca-1+CD45−TER119−) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.

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BackgroundThe specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear.ResultsTo identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs.ConclusionsA regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.