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Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. View Publication

Murine hematopoietic stem and progenitor cells (HSPCs) home to bone marrow in part by rolling on P-selectin and E-selectin expressed on endothelial cells. Human adult CD34(+) cells,which are enriched in HSPCs,roll on endothelial selectins in bone marrow vessels of nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Many human umbilical cord blood (CB) CD34(+) cells do not roll in these vessels,in part because of an uncharacterized defect in binding to P-selectin. Selectin ligands must be alpha1-3 fucosylated to form glycan determinants such as sialyl Lewis x (sLe(x)). We found that inadequate alpha1-3 fucosylation of CB CD34(+) cells,particularly CD34(+)CD38(-/low) cells that are highly enriched in HSPCs,caused them to bind poorly to E-selectin as well as to P-selectin. Treatment of CB CD34(+) cells with guanosine diphosphate (GDP) fucose and exogenous alpha1-3 fucosyltransferase VI increased cell-surface sLe(x) determinants,augmented binding to fluid-phase P- and E-selectin,and improved cell rolling on P- and E-selectin under flow. Similar treatment of CB mononuclear cells enhanced engraftment of human hematopoietic cells in bone marrows of irradiated NOD/SCID mice. These observations suggest that alpha1-3 fucosylation of CB cells might be a simple and effective method to improve hematopoietic cell homing to and engraftment in bone marrows of patients receiving CB transplants. View Publication

Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy,but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular,the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that,together with other factors,can expand mouse bone marrow HSCs in culture. Here,we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF,TPO,and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers,as assayed by NOD/SCID transplantation. A serum-free culture containing SCF,TPO,FGF-1,angiopoietin-like 5,and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs,a number potentially applicable to several clinical processes including HSC transplantation. View Publication