Human marrow stromal cells (MSCs) can be isolated from bone marrow and differentiate into multiple tissues in vitro and in vivo. These properties make them promising tools in cell and gene therapy. The lack of a specific MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion prior to clinical use. This may severely reduce MSC proliferative capacity to the point that the residual proliferative potential is insufficient to maintain long-term tissue regeneration upon reinfusion. In this study we determined the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. We report that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan.

Homing of bone marrow stromal cells (MSCs) to bone and bone marrow after transplantation, important for the correction of conditions such as metabolic storage disorders, can occur but with poor efficiency. Substantial improvements in engraftment will be required in order to derive a clinical benefit from MSC transplantation. Chemokines are the most important factors controlling cellular migration. Stromal-derived factor-1 (SDF-1) has been shown to be critical in promoting the migration of cells to the bone marrow, via its specific receptor CXCR4. The aim of our study was to investigate CXCR4 expression on MSCs and its role in mediating migration to bone marrow. We show that CXCR4, although present at the surface of a small subset of MSCs, is important for mediating specific migration of these cells to bone marrow. 4 Clinically, MSCs may be used to enhance HSC engraftment after transplantation, to correct inherited disorders of bone and cartilage, or as vehicles for gene therapy. 5,6 Crucial to the success of any of these strategies is efficient engraftment to bone and bone marrow. So far results have shown that although MSCs can engraft to those tissues, levels are at the limit of detection and clinically useful only in certain disorders such as osteogenesis imperfecta. 7 Higher levels will be required to achieve a therapeutic benefit in the majority of applications.The chemokine stromal-derived factor-1 (SDF-1) and its ligand CXCR4 play an important role in homing as shown by studies on engraftment of hematopoietic stem/progenitor cells 8 and on colonization of bone and bone marrow by metastatic breast and prostate cancer cells. 9 We have examined the expression of CXCR4 by MSCs and the migration of these cells to bone marrow stroma. Here we show for the first time that CXCR4 is important for MSC migration to bone marrow. However cell surface receptor levels are low, with large amounts found intracellularly. Study design Isolation of MSC cultures and CD34 ؉ cellsBone marrow aspirates were obtained, under local ethical approval, from the posterior iliac crest of donors aged 0 to 18 years after parental consent.MSCs were isolated by plastic adherence as previously described 10 and cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen, Paisley, United Kingdom) ϩ 10% fetal calf serum (FCS; Stem Cell Technologies, Vancouver, BC). CD34 ϩ cells were isolated using the Minimacs system according to the manufacturer's instruction (Miltenyi Biotech, Bisley, United Kingdom) Reverse-transcription-polymerase chain reaction (RT-PCR)Total RNA from MSCs or peripheral blood mononuclear cells (PBMCs), the latter activated with 10 g/mL phytohemagglutinin (PHA; Abbott Murex, Dartford, United Kingdom), was extracted using RNAzol (AMS Biotechnology, Abington, United Kingdom) according to the manufacturer's instructions, then reverse transcribed using a First Strand cDNA Synthesis kit (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). cDNA was analyzed by PCR for CXCR4 using 2 sets of primers: 668-bp amplicon forwa...

ABSTRACT+ AC133 + population was also enriched (sevenfold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133 + cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.