Rabbit Anti-Mouse Ly-6A.2 monoclonal antibody, clone 8 I5M4

Precious studies by our group showed that stromal cells from human long-term marrow cultures were mesenchymal cells following a vascular smooth muscle pathway. The present study using 58 immortalized stromal lines from different hematopoietic sites was conducted to verify whether this hypothesis also held true for murine stroma, principal components analysis performed using cytoskeletal and extracellular matrix proteins allowed the segregation of five factors explaining more than 70% of the variance. Factor I, including osteopontin and vimentin, and factor II, laminins and fibronectins, were representative of the mesenchyme. The remaining three factors mere representative of vascular smooth muscle: factor III, including alpha SM actin, SM alpha actinin, SM22 alpha, EDa(+) fibronectin, and thrombospondin-1; factor IV, metavinculin and h-caldesmon; and factor V, smooth muscle myosin SM1 and desmin. All lines expressed factors I and II; 53 lines expressed factor III, 35 lines expressed factor IV; and II lines expressed factor V. A second principal components analysis including membrane antigens indicated the cosegregration of vascular cell adhesion molecule-1 with osteopontin and that of Ly6A/E with vimentin, whereas CD34 and Thy-1 appeared to be independent factors. The heterogeneity of vascular smooth muscle markers expression suggests that harmonious maintenance of hematopoiesis depends on the cooperation between different stromal cell clones. (C) 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.

We have investigated the expression pattern of the Flk-1 receptor tyrosine kinase in mouse embryonic and fetal hematopoietic tissues as well as on hematopoietic precursor cells derived from these tissues. RNA analysis indicated that flk-1 was expressed in the yolk sac at day 10 of gestation, in the whole embryo at day 10 and 12 of gestation, in the liver throughout fetal life and in embryoid bodies (EBs) generated from ES cells differentiated in culture. Flk-1 message was also detected in erythroid and macrophage colonies generated from precursors of yolk sac, fetal liver, adult marrow and EB origin, Using an antibody directed against the extracellular portion of the molecule we have found that up to 50% of cells from EBs differentiated for 4 days express Flk-1, Following the development of this early Flk-1(+) population the number of receptor-positive cells declines progressively to represent less than 5% of the EBs by day 12 of differentiation. Kinetic analysis revealed that the establishment of the EB Flk-1(+) population precedes the development of cells which express CD34, Ly6A (Sca-1) and AA4.1. Cell sorting experiments demonstrated that all day-4 EB-derived hematopoietic precursors are Flk-1(+) whereas greater than 95% of those found within the day-12 EBs are Flk-1(-), suggesting that the precursor population which expresses this receptor represents an early but transient wave of hematopoietic development. Analysis of yolk sac and whole embryos at day 8.5 of gestation revealed a small but distinct Flk-1(+) population that contained hematopoietic precursors, Day-12.5 fetal liver contained few Flk-1(+) cells that showed little hematopoietic potential, Together these findings indicate that Flk-1 is expressed on an early population of hematopoietic precursors that may represent the onset of embryonic hematopoiesis.